The OMIT electron-density-map calculation is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase showed that the best overall results were obtained when |Fo| without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model.

Dijkstra, B.W.,

Vellieux, F.M.D.,

English

The OMIT electron-density-map calculation of Bhat [Bhat &amp; Cohen (1984). J. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21, 279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase(TM) showed that the best overall results were obtained when \F-o\ without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model.</p

Vellieux, FMD

Dijkstra, BW

ARTS repository - University of Groningen

   University of GroningenComputation of Bhat's OMIT maps with different coefficientsVellieux, FMD; Dijkstra, BWPublished in:Journal of Applied CrystallographyDOI:10.1107/S0021889896012551IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vellieux, FMD., & Dijkstra, BW. (1997). Computation of Bhat's OMIT maps with different coefficients.Journal of Applied Crystallography, 30(3), 396-399. https://doi.org/10.1107/S0021889896012551CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 07-06-2022396 SHORT C O M M U N I C A T I O N S  J Appl. Cryst. (1997). 30, 396-399 C o m p u t a t i o n  o f  Bhat ' s  O M I T  m a p s  wi th  d i f ferent  coeff ic ients  E M. D. VELLIEUX a AND B. W. DIJKSTRA b at %aboratoire de Cristallographie et de CristallogenOse des Prot~ines, Institut de Biologie Structurale Jean-Pierre Ebel CEA CNRS, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 01, France, and bBIOSON Research Institute and Laboratory of  Biophysical Chemistry, State University of  Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: vellieux@ibs.ibs.fr (Received 5 August 1996; accepted 7 October 1996) Abstract The OMIT electron-density-map calculation of Bhat [Bhat & Cohen (1984)..I. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21,279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase ~ showed that the best overall results were obtained when ]Fol without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model. 1. Introduction The OMIT electron-density maps computed using Bhat's procedure (Bhat & Cohen, 1984; Bhat, 1988) are known to be very effective for the discovery of errors in the crystal structure determination of macromolecules. However, in their publications, Bhat and coauthor have not investigated the effects of different Fourier coefficients on the quality of the resulting OMIT maps. The subject of this paper is the study of several types of amplitude coefficients for the computation of the initial electron-density distribution from which OMIT maps are calculated, and the analysis of the resulting maps. 2. Definitions IFol and IFcl are the observed and calculated structure-factor amplitudes, respectively, and IEol is the normalized structure- factor amplitude for the observed reflection, m is Sim's figure of merit, m = 1 I(X)/Io(X ) for acentric reflections and m = tanh(X/2), where 11 and I 0 are the modified Bessel functions of order 1 and 0, respectively, and X = 2lEo liE c I /E L (~L = EF,~) SIGMAA: Read (1986) has shown that electron-density maps calculated with amplitude coefficients of the form 2mlFol-  DIF~I for acentric reflections and mlFol for centric reflections are less biased towards the macromolecular model from which the IFcl's are calculated. In this work, we have used modified SIGMAA coefficients of the form 3mlFol - 2Olfcl for acentric reflections and 2mlFol-  DIF~I for centric reflections (vide infra). OMIT maps: the reader is referred to Bhat (1988) for a complete description of the reciprocal-space procedure used to compute such maps, in which the electron density of a 'phasing volume' plus that of a 'neutral volume' surrounding it do not contribute to the IFcl's and $c's used in the procedure. CCF 1 is a real-space correlation coefficient with respect to a 'control map', without any resolution cut-off, computed from refined atomic coordinates. CCF2 is a correlation coefficient between the OMIT map and a 2.0A-resolution artificial electron-density distribution generated from refined atomic coordinates. 3. Material and methods A Fortran program called OMITwas written that allows one to compute Bhat's OMIT maps in a single step. This program requires amplitudes and phases in the CCP4 (Collaborative Computational Project, Number 4, 1994) MTZ format, and generates an OMIT map in the Groningen BIOMOL Master Fourier Map (MFF) format, which can be converted to other formats (e.g. DSN6, CCP4, X-PLOR) using separate programs that are available from the authors. In this study, we have used 'classical' coefficients of the form Eo, Fo, mFo, 2F o - F  c etc. (Table 1) to compute the initial electron-density distribution from which Bhat's OMIT maps are calculated. Concomitantly, we have developed a new type of computation of OMIT maps: in the first step, a 'classical' electron-density map is computed and the histogram of densities (Lunin, 1988; Zhang & Main, 1990) of this map is generated. The OMIT map is then computed and, finally, the histogram of densities of the OMIT map is adjusted to fit that of the initial electron-density map. All our tests have been carried out using models of the structure of monoclinic Savinase ~ in complex with the active site inhibitor benzo-furan boronic acid (BCsO3HT) (Verger, Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997). We used the 2.0 A-resolution unrefined model for the computation of the calculated amplitudes and phases used in the OMIT-map calculations. For the computation of the real-space correlation coefficient CCF1 between the observed OMIT density and the calculated density generated from the refined molecular model, carried out with the program. O (Jones, Zou, Cowan & Kjeldgaard, 1991), the 1.7A-resolution refined model of Savinase ~ was used to generate a calculated density with no resolution cut-off. We also computed, using the program MAPMAN (Kleywegt & Jones, 1996), the correlation coeffi- cient CCF2 between the OMIT maps and a 2.0 ,~-resolution artificial electron-density distribution generated from refined atomic coordinates. Savinase ~ crystallizes in space group P21, with cell dimensions a = 40.83, b = 72.91 and c - -  76.27 ,~, and ~ = 90.0, fl = 91.21 amd ~/= 90.0 °. The crystals contain two molecules of Savinase @ in the asymmetric unit. For the OMIT- map calculations, the asymmetric unit was divided into 30 phased volumes, each surrounded by a neutral volume © 1997 Intemational Union of Crystallography Printed in Great Britain - all fights reserved Journal of Applied Crystallography ISSN 0021-8898 © 1997 SHORT COMMUNICATIONS 397 ,0068, (a) (b) (c) ( d )  (e) Fig. 1. OMIT electron-density maps, computed with different initial amplitude coefficients. Contrary to the electron densities usually shown, this figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. (a) 2.0 ~,-resolution OMIT map computed with initial amplitude coefficients of  the form 3 [Fo[ - 2[Fc[ and phases from the unrefined model of Savinase @. (b) As (a), with initial amplitude coefficients of the form [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), with modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - D[Fc[ for the acentric reflections. (e) 1.7 ~,-resolution OMIT map computed with modified SIGMAA initial amplitude coefficients and phases from the refined model of Savinase @. This map w.as computed using the prerelease of  X-PLORVers ion  4.0 (Briinger, personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model of Savinase ~ before refinement; thick lines: 1.7 ,~-resolution model of Savinase ~ after refinement with X-PLOR.  All maps were displayed using the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) and are contoured at the 1.0a level. 398 SHORT C O M M U N I C A T I O N S  Table 1. Summary o f  the results obtained with several amplitude coefficients for  the computation o f  the initial electron-density distribution from which the OMIT map is calculated HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- density distribution obtained from refined atomic coordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients are shown in bold. With HM Without HM Coefficient Min CCF1 Max CCFI (CCFI) Min CCFI Max CCFI (CCF1) CCF2 IEol 0.390 0.852 0.659 0.390 0.849 0.663 0.478 IFol 0.380 0.856 0.663 0.396 0.853 0.669 0.479 mlFol 0.348 0.844 0.643 0.348 0.844 0.644 0.454 21Fol - IFcl 0.351 0.859 0.652 0.351 0.858 0.651 0.273 m(2lFol- IFcl) 0.328 0.850 0.637 0.329 0.850 0.637 0.439 3 IFol - 2lFcl 0.252 0.837 0.605 0.251 0.838 0.604 0.395 m(31Fol - 21F¢1) 0.255 0.841 0.622 0.257 0.843 0.621 0.416 3mlFol - 2DIFcl 0.349 0.862 0.644 0.332 0.864 0.638 0.449 m 0.413 0.827 0.653 0.416 0.826 0.657 0.469 consisting of two grid points in each direction (terms are defined as in Bhat, 1988). Since simulated-annealing OMIT- map calculations (Hodel, Kim & Briinger, 1992) took exceedingly long to compute (so long that such a computation was not completed after more than 24 h CPU time on our three- processor DEC ALPHA server), we did not investigate the effect of  such refinement procedures together with different initial amplitude coefficients on the quality of  the resulting OMIT maps. These OMIT maps, including the step of  histogram mapping, may now be computed using the prerelease of  X-PLOR Version 4.0 (Briinger, personal communication; Hodel, Kim & Briinger, 1992; Briinger, Kuriyan & Karplus, 1987). 4. Results Our results (Table 1 and Fig. 1) can be summarized as follows: (i) Resetting the electron density values less than - 1 . 0  times the root-mean-square (r.m.s.) electron density to - 1 . 0  times the r.m.s, electron density, as suggested by Bhat (1988), gave poorer OMIT maps than those computed with omission of  this step (data not shown). Hence, in all our tests, this tnmcation step was omitted, so that Ifml = Ifcl, Wf = 1.0 and W = W, for all reflections (these terms are defined as in Bhat, 1988). Attempts have not been made to vary the threshold or the resolution of the data to analyse the usefulness of  non- negativity criteria in OMIT maps. (ii) The highest correlations between the OMIT map and the calculated maps from the refined molecular model are obtained when no figure-of-merit (Sim, 1959, 1960) scheme is used, except for the OMIT maps computed with initial 31/7o[ - 2[Fc[ amplitude coefficients and for modified SIGMAA initial amplitude coefficients of  the form 3m[Fo[ - 2D[F~[ for acentric reflections and 2m[Fo[-D[F~[ for centric reflections (the modified SIGMAA coefficients give improved electron-density maps with respect to the 'classical' coefficients of  the form 2mlFo l -  D[Fc[ for acentric reflections and m[Fol for centric reflections: because the crystallographic refinement problem is undetermined for protein structures, where we are usually far from atomic resolution, it is possible to overfit the amplitudes in structure refinement. The amplitude differences are therefore smaller than they would be if the errors in the structure were truly random. To correct for that effect, in part, we can scale up the contribution of  the differences in the electron-density-map coefficients (Vellieux, unpublished work; Read, 1986; Vijayan, 1980). (iii) The effect of  adjusting the histogram of densities of  the OMIT map to fit that of  the initial map is negligible. This is perhaps not surprising a posteriori, since the application of histogram modification is important only for the real-space correlation-coefficient calculation and not for the map quality, since it is simply a nonlinear rescaling of the map and influences only the choice of  the isoline level. (iv) The most surprising result has been that, in the regions of  the map where the model is incorrect, with a low real-space correlation coefficient, very interesting OMIT maps (minimum correlation 0.416) are obtained when only the Sim figure-of- merit is used as the initial map amplitude coefficient (Fig. 1 c). This indicates, once again, that phases are more important than structure-factor amplitudes when it comes to the calculation of an electron-density distribution. Thus, these tests suggest that this type of  OMIT map, together with OMIT maps computed with IFol and with modified SIGMAA coefficients as the initial amplitude, are particularly useful to reconstruct the macro- molecular model in the grossly incorrect regions of  the model. It has not escaped our attention that such OMIT maps may also be used as the starting point for electron-density modification procedures. Tests are currently being carried out using the DEMON~ANGEL density-modification software suite (Vellieux, Hunt, Roy & Read, 1995) to investigate such OMIT maps in this context. It can also be suggested, from the results summarized in Table 1, that the use of  several OMIT maps, computed with different initial amplitude coefficients (i.e. only the Sim figure of  merit m, IFol and modified SIGMAA coefficients of  the form 3m IFol - 2DIFcI for acentric reflections and 2mlFol - DIFcI for centric reflections) may be an optimal strategy in the detection of  errors in a macromolecular model. The OMIT program is available freely upon request to the authors (e-mail vellieux@ibs.ibs.fr or bauke@chem.rug.nl). S H O R T  C O M M U N I C A T I O N S  399 The members of the lnstitut de Biologie Structurale Jean- Pierre Ebel CEA CNRS dedicate this paper to the memory of Rosaria and Serge Pares, who died on board TWA flight 800 off the Long Island coast. We thank Professor A. T. Briinger (Yale University) for having provided the prerelease of X-PLOR Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading of  the manuscript. Financial support of the French CEA and CNRS, and of the Netherlands Foundation for Chemical Research (SON), is gratefully acknowledged. References Bhat, T. N & Cohen, G. H (1984). J Appl. Cryst. 17, 244-248. Bhat, T. N. (1988). J Appl. Cryst. 21,279-281. Briinger, A. T., Kuriyan, J. & Karplus, M. (1987). Science, 235, 458-460. Hodel, A., Kim, S.-H. & Briinger, A. T. (1992). Acta Cryst. A48, 85 i-859. Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110-119. Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst. D52, 826-828. Lunin, V. Y. (1988). Acta Cryst. A44, 144-150. Read, R. J. (1986). Acta Cryst. A42, 140-149. Sim, G. A. (1959). Acta Cryst. 12, 813-815. Sim, G. A. (1960). Acta Cryst. 13, 511-512. Vellieux, E M. D. A. P., Hunt, J. F., Roy, S. & Read, R. J. (1995). J. Appl. Cryst. 28, 347-351. Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, J. C. (1997). In preparation. Vijayan, M. (1980). Acta Cryst. A36, 295-298. Zhang, K. Y. J. & Main, P. (1990). Acta Cryst. A46, 41-46. 

Computation of Bhat's OMIT maps with different coefficients

F. M. D. Vellieux

B. W. Dijkstra

Crossref

Journal of Applied Crystallography

University of Groningen Digital Archive

396  SHORT COMMUNICATIONS 
J  Appl.  Cryst.  (1997).  30, 396-399 
Computation  of Bhat's  OMIT  maps  with  different  coefficients 
E  M.  D. VELLIEUX  a AND B. W. DIJKSTRA  b at %aboratoire de Cristallographie et de CristallogenOse  des Prot~ines,  Institut de 
Biologie Structurale Jean-Pierre Ebel CEA CNRS,  41 Avenue des Martyrs,  38027 Grenoble CEDEX 01, France,  and bBIOSON 
Research Institute and Laboratory of Biophysical Chemistry,  State University of Groningen,  Nijenborgh 4,  9747 AG Groningen, 
The Netherlands. E-mail: vellieux@ibs.ibs.fr 
(Received 5 August 1996; accepted 7 October 1996) 
Abstract 
The  OMIT electron-density-map  calculation of Bhat  [Bhat & 
Cohen  (1984)..I.  Appl.  Cryst.  17,  244-248;  Bhat  (1988).  J. 
Appl. Cryst. 21,279-281]  is very effective in discovering errors 
in  a  macromolecular  structure  determination.  A  Fortran 
program called OMIT has been written to calculate such maps 
and an investigation has been carried out into which coefficients 
for the map calculation produce the best OMIT maps. Testing of 
the program on Savinase  ~  showed that the best overall results 
were obtained when  ]Fol without figure of merit was used.  In 
regions where the map is incorrect, the most interesting OMIT 
maps are produced  when only the figure of merit, or modified 
SIGMAA  coefficients,  are  used  as  the  initial  map  amplitude 
coefficients. Thus, these tests suggest that such OMIT maps are 
particularly useful to reconstruct the macromolecular model in 
the grossly incorrect regions of the model. 
1. Introduction 
The  OMIT  electron-density  maps  computed  using  Bhat's 
procedure  (Bhat  &  Cohen,  1984;  Bhat,  1988)  are  known  to 
be  very  effective  for  the  discovery  of errors  in  the  crystal 
structure  determination  of macromolecules.  However,  in their 
publications,  Bhat  and  coauthor  have  not  investigated  the 
effects  of different  Fourier  coefficients  on  the  quality of the 
resulting OMIT maps. The subject of this paper is the study of 
several types of amplitude coefficients for the computation  of 
the initial electron-density distribution from which OMIT maps 
are calculated, and the analysis of the resulting maps. 
2. Definitions 
IFol and  IFcl are the observed  and  calculated  structure-factor 
amplitudes,  respectively, and  IEol is the normalized structure- 
factor amplitude for the observed reflection, m is Sim's figure of 
merit,  m = 1  I(X)/Io(X  )  for  acentric  reflections  and 
m =  tanh(X/2),  where  11  and  I 0  are  the  modified  Bessel 
functions of order 1 and 0, respectively, and X  =  2lEo liE  c I/E  L 
(~L =  EF,~) 
SIGMAA: Read (1986) has shown that electron-density maps 
calculated  with  amplitude  coefficients  of  the  form 
2mlFol- DIF~I for  acentric  reflections  and  mlFol  for  centric 
reflections are  less biased towards  the macromolecular  model 
from which the IFcl's are calculated. In this work, we have used 
modified SIGMAA coefficients of the form 3mlFol -  2Olfcl for 
acentric reflections and  2mlFol- DIF~I for centric  reflections 
(vide infra). 
OMIT  maps:  the  reader  is  referred  to  Bhat  (1988)  for  a 
complete description of the reciprocal-space procedure used to 
compute such maps, in which the electron density of a 'phasing 
volume'  plus that of a  'neutral volume' surrounding it do not 
contribute to the IFcl's and $c's used in the procedure. 
CCF 1 is a real-space correlation coefficient with respect to a 
'control map',  without any resolution cut-off,  computed  from 
refined  atomic  coordinates.  CCF2  is  a  correlation  coefficient 
between  the  OMIT  map  and  a  2.0A-resolution  artificial 
electron-density  distribution  generated  from  refined  atomic 
coordinates. 
3. Material and methods 
A  Fortran program called OMITwas written that allows one to 
compute  Bhat's  OMIT  maps  in  a  single  step.  This  program 
requires  amplitudes  and  phases  in  the  CCP4  (Collaborative 
Computational  Project,  Number  4,  1994)  MTZ  format,  and 
generates  an  OMIT  map  in the  Groningen  BIOMOL  Master 
Fourier Map  (MFF)  format,  which can be  converted to  other 
formats (e.g. DSN6, CCP4, X-PLOR) using separate programs 
that are available from the authors. 
In this study, we have used 'classical' coefficients of the form 
Eo,  Fo,  mFo,  2F  o -F c  etc.  (Table  1)  to  compute  the  initial 
electron-density distribution from which Bhat's OMIT maps are 
calculated.  Concomitantly, we  have developed  a  new  type  of 
computation  of OMIT  maps:  in  the  first  step,  a  'classical' 
electron-density  map  is  computed  and  the  histogram  of 
densities (Lunin,  1988;  Zhang &  Main,  1990)  of this map  is 
generated.  The OMIT map  is then computed  and,  finally, the 
histogram of densities of the OMIT map is adjusted to fit that of 
the initial electron-density map. 
All  our  tests  have  been  carried  out  using  models  of the 
structure  of monoclinic Savinase  ~  in complex with the active 
site  inhibitor  benzo-furan  boronic  acid  (BCsO3HT)  (Verger, 
Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997).  We used 
the 2.0 A-resolution unrefined model for the computation of the 
calculated  amplitudes  and  phases  used  in  the  OMIT-map 
calculations.  For the computation of the real-space correlation 
coefficient CCF1  between the observed OMIT density and the 
calculated density generated from the refined molecular model, 
carried  out  with  the  program.  O  (Jones,  Zou,  Cowan  & 
Kjeldgaard,  1991),  the  1.7A-resolution  refined  model  of 
Savinase  ~  was used to generate  a  calculated density with no 
resolution  cut-off.  We  also  computed,  using  the  program 
MAPMAN (Kleywegt &  Jones,  1996),  the  correlation  coeffi- 
cient  CCF2  between  the  OMIT  maps  and  a  2.0 ,~-resolution 
artificial  electron-density  distribution  generated  from  refined 
atomic coordinates. 
Savinase  ~  crystallizes  in  space  group  P21,  with  cell 
dimensions a  =  40.83,  b =  72.91  and  c--  76.27 ,~,  and  ~ = 
90.0,  fl =  91.21  amd  ~/=  90.0 °.  The  crystals  contain  two 
molecules of Savinase  @ in the asymmetric unit. For the OMIT- 
map  calculations,  the  asymmetric  unit  was  divided  into  30 
phased  volumes,  each  surrounded  by  a  neutral  volume 
©  1997 Intemational Union of Crystallography 
Printed in Great Britain -  all fights reserved 
Journal of Applied Crystallography 
ISSN 0021-8898  ©  1997 SHORT COMMUNICATIONS  397 
,0068, 
(a)  (b)  (c) 
(d)  (e) 
Fig.  1. OMIT electron-density maps, computed with different initial  amplitude coefficients. Contrary to the electron densities usually shown, this 
figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is 
evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density 
suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two 
conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. 
(a) 2.0 ~,-resolution OMIT map computed with initial  amplitude coefficients of the form 3 [Fo[ -  2[Fc[ and phases from the unrefined model of 
Savinase  @. (b) As (a), with initial amplitude coefficients of the form  [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), 
with modified SIGMAA  initial  amplitude coefficients of the form 3m[Fo[  -  D[Fc[  for the acentric reflections. (e) 1.7 ~,-resolution OMIT map 
computed with modified SIGMAA  initial amplitude coefficients and phases from the refined model of Savinase  @. This map w.as computed using 
the prerelease of X-PLORVersion 4.0 (Briinger,  personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model 
of Savinase  ~  before refinement; thick lines:  1.7 ,~-resolution model of Savinase  ~  after refinement with X-PLOR. All maps were displayed using 
the program O (Jones, Zou, Cowan & Kjeldgaard,  1991) and are contoured at the 1.0a level. 398  SHORT  COMMUNICATIONS 
Table  1. Summary of the results obtained with several amplitude coefficients for the computation  of the initial electron-density 
distribution from which the OMIT map is calculated 
HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), 
except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space 
correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- 
density distribution obtained from refined atomic coordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a 
residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation 
coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- 
resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients 
are shown in bold. 
With HM  Without HM 
Coefficient  Min CCF1  Max CCFI  (CCFI)  Min CCFI  Max CCFI  (CCF1)  CCF2 
IEol  0.390  0.852  0.659  0.390  0.849  0.663  0.478 
IFol  0.380  0.856  0.663  0.396  0.853  0.669  0.479 
mlFol  0.348  0.844  0.643  0.348  0.844  0.644  0.454 
21Fol -  IFcl  0.351  0.859  0.652  0.351  0.858  0.651  0.273 
m(2lFol- IFcl)  0.328  0.850  0.637  0.329  0.850  0.637  0.439 
3 IFol -  2lFcl  0.252  0.837  0.605  0.251  0.838  0.604  0.395 
m(31Fol -  21F¢1)  0.255  0.841  0.622  0.257  0.843  0.621  0.416 
3mlFol -  2DIFcl  0.349  0.862  0.644  0.332  0.864  0.638  0.449 
m  0.413  0.827  0.653  0.416  0.826  0.657  0.469 
consisting  of  two  grid  points  in  each  direction  (terms  are 
defined as  in  Bhat,  1988).  Since  simulated-annealing OMIT- 
map  calculations  (Hodel,  Kim  &  Briinger,  1992)  took 
exceedingly long to compute (so long that such a computation 
was not completed after more than 24 h CPU time on our three- 
processor  DEC  ALPHA  server),  we  did  not  investigate  the 
effect  of such  refinement procedures  together  with  different 
initial  amplitude  coefficients  on  the  quality  of the  resulting 
OMIT  maps.  These  OMIT  maps,  including  the  step  of 
histogram mapping, may now be computed using the prerelease 
of X-PLOR  Version  4.0  (Briinger,  personal  communication; 
Hodel,  Kim &  Briinger,  1992;  Briinger,  Kuriyan  &  Karplus, 
1987). 
4.  Results 
Our results (Table  1 and Fig.  1) can be summarized as follows: 
(i) Resetting the electron density values less than -1.0 times 
the root-mean-square (r.m.s.) electron density to -1.0 times the 
r.m.s,  electron  density,  as  suggested  by  Bhat  (1988),  gave 
poorer OMIT maps than those computed with omission of this 
step  (data not shown).  Hence, in all  our tests,  this tnmcation 
step was omitted, so that  Ifml =  Ifcl,  Wf =  1.0  and  W =  W, 
for all  reflections (these terms are  defined as  in Bhat,  1988). 
Attempts  have  not  been  made  to  vary  the  threshold  or  the 
resolution  of  the  data  to  analyse  the  usefulness  of  non- 
negativity criteria in OMIT maps. 
(ii) The highest correlations between the OMIT map and the 
calculated maps from the refined molecular model are obtained 
when  no  figure-of-merit (Sim,  1959,  1960)  scheme  is  used, 
except for the OMIT maps computed with initial 31/7o[ -  2[Fc[ 
amplitude  coefficients  and  for  modified  SIGMAA  initial 
amplitude coefficients of the form 3m[Fo[ -  2D[F~[  for acentric 
reflections  and  2m[Fo[-D[F~[  for  centric  reflections  (the 
modified SIGMAA  coefficients give improved electron-density 
maps  with  respect  to  the  'classical'  coefficients of the  form 
2mlFol- D[Fc[ for  acentric  reflections and  m[Fol  for  centric 
reflections: because the crystallographic refinement problem is 
undetermined for protein structures,  where we are  usually far 
from atomic resolution, it is possible to overfit the amplitudes in 
structure  refinement. The  amplitude differences are  therefore 
smaller than they would be if the errors in the structure were 
truly random. To correct for that effect, in part, we can scale up 
the contribution of the differences in the electron-density-map 
coefficients (Vellieux, unpublished work; Read,  1986; Vijayan, 
1980). 
(iii) The effect of adjusting the histogram of densities of the 
OMIT map to fit that of the initial map  is negligible. This  is 
perhaps  not  surprising  a  posteriori,  since  the  application  of 
histogram  modification  is  important  only  for  the  real-space 
correlation-coefficient calculation and not for the map quality, 
since  it  is  simply  a  nonlinear  rescaling  of  the  map  and 
influences only the choice of the isoline level. 
(iv) The most surprising result has been that, in the regions of 
the map  where the model is  incorrect,  with  a  low real-space 
correlation coefficient, very interesting OMIT maps (minimum 
correlation 0.416)  are  obtained when only the  Sim figure-of- 
merit is used as the initial map amplitude coefficient (Fig.  1  c). 
This indicates, once again, that phases are more important than 
structure-factor amplitudes when it comes to the calculation of 
an electron-density distribution. Thus,  these tests suggest that 
this type of OMIT map, together with OMIT maps computed 
with IFol and with modified SIGMAA  coefficients as the initial 
amplitude,  are  particularly  useful  to  reconstruct  the  macro- 
molecular model in the grossly incorrect regions of the model. 
It has not escaped our attention that such OMIT maps may 
also  be  used  as  the  starting  point  for  electron-density 
modification procedures. Tests  are currently being carried out 
using the DEMON~ANGEL density-modification software suite 
(Vellieux, Hunt, Roy &  Read,  1995) to investigate such OMIT 
maps in this context. It can also be suggested, from the results 
summarized in Table  1,  that  the use  of several  OMIT  maps, 
computed with different initial amplitude coefficients (i.e.  only 
the  Sim  figure  of  merit  m,  IFol and  modified  SIGMAA 
coefficients of the form 3m IFol -  2DIFcI for acentric reflections 
and 2mlFol -  DIFcI  for centric reflections) may be an optimal 
strategy in the detection of errors in a macromolecular model. 
The  OMIT program  is  available  freely upon request to the 
authors (e-mail vellieux@ibs.ibs.fr or bauke@chem.rug.nl). SHORT  COMMUNICATIONS  399 
The  members  of the  lnstitut  de  Biologie  Structurale  Jean- 
Pierre Ebel CEA CNRS dedicate this paper to the memory of 
Rosaria and Serge Pares, who died on board TWA flight 800 off 
the Long Island coast. We thank Professor A. T. Briinger (Yale 
University)  for  having  provided  the  prerelease  of  X-PLOR 
Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading 
of the  manuscript.  Financial  support  of the  French CEA  and 
CNRS,  and  of  the  Netherlands  Foundation  for  Chemical 
Research (SON),  is gratefully acknowledged. 
References 
Bhat, T. N & Cohen, G. H (1984). J  Appl.  Cryst.  17, 244-248. 
Bhat, T. N. (1988). J  Appl.  Cryst.  21,279-281. 
Briinger,  A.  T.,  Kuriyan,  J.  &  Karplus,  M.  (1987). Science,  235, 
458-460. 
Hodel,  A.,  Kim,  S.-H.  &  Briinger,  A.  T.  (1992).  Acta  Cryst.  A48, 
85 i-859. 
Collaborative  Computational  Project,  Number 4  (1994).  Acta  Cryst. 
D50, 760-763. 
Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta 
Cryst.  A47,  110-119. 
Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst.  D52, 826-828. 
Lunin, V. Y. (1988). Acta Cryst.  A44,  144-150. 
Read, R. J. (1986). Acta Cryst.  A42,  140-149. 
Sim, G. A. (1959). Acta Cryst.  12, 813-815. 
Sim, G. A. (1960). Acta Cryst.  13, 511-512. 
Vellieux, E  M. D. A. P., Hunt, J.  F., Roy, S. &  Read, R. J. (1995). J. 
Appl.  Cryst.  28, 347-351. 
Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, 
J. C. (1997). In preparation. 
Vijayan, M. (1980). Acta Cryst.  A36, 295-298. 
Zhang, K. Y. J. & Main, P. (1990). Acta Cryst.  A46, 41-46. 

Acta Cryst.

Dissertations of the University of Groningen

   University of GroningenComputation of Bhat's OMIT maps with different coefficientsVellieux, FMD; Dijkstra, BWPublished in:Journal of Applied CrystallographyDOI:10.1107/S0021889896012551IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vellieux, FMD., & Dijkstra, BW. (1997). Computation of Bhat's OMIT maps with different coefficients.Journal of Applied Crystallography, 30(3), 396-399. https://doi.org/10.1107/S0021889896012551CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 17-07-2023396 SHORT C O M M U N I C A T I O N S  J Appl. Cryst. (1997). 30, 396-399 C o m p u t a t i o n  o f  Bhat ' s  O M I T  m a p s  wi th  d i f ferent  coeff ic ients  E M. D. VELLIEUX a AND B. W. DIJKSTRA b at %aboratoire de Cristallographie et de CristallogenOse des Prot~ines, Institut de Biologie Structurale Jean-Pierre Ebel CEA CNRS, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 01, France, and bBIOSON Research Institute and Laboratory of  Biophysical Chemistry, State University of  Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: vellieux@ibs.ibs.fr (Received 5 August 1996; accepted 7 October 1996) Abstract The OMIT electron-density-map calculation of Bhat [Bhat & Cohen (1984)..I. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21,279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase ~ showed that the best overall results were obtained when ]Fol without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model. 1. Introduction The OMIT electron-density maps computed using Bhat's procedure (Bhat & Cohen, 1984; Bhat, 1988) are known to be very effective for the discovery of errors in the crystal structure determination of macromolecules. However, in their publications, Bhat and coauthor have not investigated the effects of different Fourier coefficients on the quality of the resulting OMIT maps. The subject of this paper is the study of several types of amplitude coefficients for the computation of the initial electron-density distribution from which OMIT maps are calculated, and the analysis of the resulting maps. 2. Definitions IFol and IFcl are the observed and calculated structure-factor amplitudes, respectively, and IEol is the normalized structure- factor amplitude for the observed reflection, m is Sim's figure of merit, m = 1 I(X)/Io(X ) for acentric reflections and m = tanh(X/2), where 11 and I 0 are the modified Bessel functions of order 1 and 0, respectively, and X = 2lEo liE c I /E L (~L = EF,~) SIGMAA: Read (1986) has shown that electron-density maps calculated with amplitude coefficients of the form 2mlFol-  DIF~I for acentric reflections and mlFol for centric reflections are less biased towards the macromolecular model from which the IFcl's are calculated. In this work, we have used modified SIGMAA coefficients of the form 3mlFol - 2Olfcl for acentric reflections and 2mlFol-  DIF~I for centric reflections (vide infra). OMIT maps: the reader is referred to Bhat (1988) for a complete description of the reciprocal-space procedure used to compute such maps, in which the electron density of a 'phasing volume' plus that of a 'neutral volume' surrounding it do not contribute to the IFcl's and $c's used in the procedure. CCF 1 is a real-space correlation coefficient with respect to a 'control map', without any resolution cut-off, computed from refined atomic coordinates. CCF2 is a correlation coefficient between the OMIT map and a 2.0A-resolution artificial electron-density distribution generated from refined atomic coordinates. 3. Material and methods A Fortran program called OMITwas written that allows one to compute Bhat's OMIT maps in a single step. This program requires amplitudes and phases in the CCP4 (Collaborative Computational Project, Number 4, 1994) MTZ format, and generates an OMIT map in the Groningen BIOMOL Master Fourier Map (MFF) format, which can be converted to other formats (e.g. DSN6, CCP4, X-PLOR) using separate programs that are available from the authors. In this study, we have used 'classical' coefficients of the form Eo, Fo, mFo, 2F o - F  c etc. (Table 1) to compute the initial electron-density distribution from which Bhat's OMIT maps are calculated. Concomitantly, we have developed a new type of computation of OMIT maps: in the first step, a 'classical' electron-density map is computed and the histogram of densities (Lunin, 1988; Zhang & Main, 1990) of this map is generated. The OMIT map is then computed and, finally, the histogram of densities of the OMIT map is adjusted to fit that of the initial electron-density map. All our tests have been carried out using models of the structure of monoclinic Savinase ~ in complex with the active site inhibitor benzo-furan boronic acid (BCsO3HT) (Verger, Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997). We used the 2.0 A-resolution unrefined model for the computation of the calculated amplitudes and phases used in the OMIT-map calculations. For the computation of the real-space correlation coefficient CCF1 between the observed OMIT density and the calculated density generated from the refined molecular model, carried out with the program. O (Jones, Zou, Cowan & Kjeldgaard, 1991), the 1.7A-resolution refined model of Savinase ~ was used to generate a calculated density with no resolution cut-off. We also computed, using the program MAPMAN (Kleywegt & Jones, 1996), the correlation coeffi- cient CCF2 between the OMIT maps and a 2.0 ,~-resolution artificial electron-density distribution generated from refined atomic coordinates. Savinase ~ crystallizes in space group P21, with cell dimensions a = 40.83, b = 72.91 and c - -  76.27 ,~, and ~ = 90.0, fl = 91.21 amd ~/= 90.0 °. The crystals contain two molecules of Savinase @ in the asymmetric unit. For the OMIT- map calculations, the asymmetric unit was divided into 30 phased volumes, each surrounded by a neutral volume © 1997 Intemational Union of Crystallography Printed in Great Britain - all fights reserved Journal of Applied Crystallography ISSN 0021-8898 © 1997 SHORT COMMUNICATIONS 397 ,0068, (a) (b) (c) ( d )  (e) Fig. 1. OMIT electron-density maps, computed with different initial amplitude coefficients. Contrary to the electron densities usually shown, this figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. (a) 2.0 ~,-resolution OMIT map computed with initial amplitude coefficients of  the form 3 [Fo[ - 2[Fc[ and phases from the unrefined model of Savinase @. (b) As (a), with initial amplitude coefficients of the form [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), with modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - D[Fc[ for the acentric reflections. (e) 1.7 ~,-resolution OMIT map computed with modified SIGMAA initial amplitude coefficients and phases from the refined model of Savinase @. This map w.as computed using the prerelease of  X-PLORVers ion  4.0 (Briinger, personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model of Savinase ~ before refinement; thick lines: 1.7 ,~-resolution model of Savinase ~ after refinement with X-PLOR.  All maps were displayed using the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) and are contoured at the 1.0a level. 398 SHORT C O M M U N I C A T I O N S  Table 1. Summary o f  the results obtained with several amplitude coefficients for  the computation o f  the initial electron-density distribution from which the OMIT map is calculated HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- density distribution obtained from refined atomic coordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients are shown in bold. With HM Without HM Coefficient Min CCF1 Max CCFI (CCFI) Min CCFI Max CCFI (CCF1) CCF2 IEol 0.390 0.852 0.659 0.390 0.849 0.663 0.478 IFol 0.380 0.856 0.663 0.396 0.853 0.669 0.479 mlFol 0.348 0.844 0.643 0.348 0.844 0.644 0.454 21Fol - IFcl 0.351 0.859 0.652 0.351 0.858 0.651 0.273 m(2lFol- IFcl) 0.328 0.850 0.637 0.329 0.850 0.637 0.439 3 IFol - 2lFcl 0.252 0.837 0.605 0.251 0.838 0.604 0.395 m(31Fol - 21F¢1) 0.255 0.841 0.622 0.257 0.843 0.621 0.416 3mlFol - 2DIFcl 0.349 0.862 0.644 0.332 0.864 0.638 0.449 m 0.413 0.827 0.653 0.416 0.826 0.657 0.469 consisting of two grid points in each direction (terms are defined as in Bhat, 1988). Since simulated-annealing OMIT- map calculations (Hodel, Kim & Briinger, 1992) took exceedingly long to compute (so long that such a computation was not completed after more than 24 h CPU time on our three- processor DEC ALPHA server), we did not investigate the effect of  such refinement procedures together with different initial amplitude coefficients on the quality of  the resulting OMIT maps. These OMIT maps, including the step of  histogram mapping, may now be computed using the prerelease of  X-PLOR Version 4.0 (Briinger, personal communication; Hodel, Kim & Briinger, 1992; Briinger, Kuriyan & Karplus, 1987). 4. Results Our results (Table 1 and Fig. 1) can be summarized as follows: (i) Resetting the electron density values less than - 1 . 0  times the root-mean-square (r.m.s.) electron density to - 1 . 0  times the r.m.s, electron density, as suggested by Bhat (1988), gave poorer OMIT maps than those computed with omission of  this step (data not shown). Hence, in all our tests, this tnmcation step was omitted, so that Ifml = Ifcl, Wf = 1.0 and W = W, for all reflections (these terms are defined as in Bhat, 1988). Attempts have not been made to vary the threshold or the resolution of the data to analyse the usefulness of  non- negativity criteria in OMIT maps. (ii) The highest correlations between the OMIT map and the calculated maps from the refined molecular model are obtained when no figure-of-merit (Sim, 1959, 1960) scheme is used, except for the OMIT maps computed with initial 31/7o[ - 2[Fc[ amplitude coefficients and for modified SIGMAA initial amplitude coefficients of  the form 3m[Fo[ - 2D[F~[ for acentric reflections and 2m[Fo[-D[F~[ for centric reflections (the modified SIGMAA coefficients give improved electron-density maps with respect to the 'classical' coefficients of  the form 2mlFo l -  D[Fc[ for acentric reflections and m[Fol for centric reflections: because the crystallographic refinement problem is undetermined for protein structures, where we are usually far from atomic resolution, it is possible to overfit the amplitudes in structure refinement. The amplitude differences are therefore smaller than they would be if the errors in the structure were truly random. To correct for that effect, in part, we can scale up the contribution of  the differences in the electron-density-map coefficients (Vellieux, unpublished work; Read, 1986; Vijayan, 1980). (iii) The effect of  adjusting the histogram of densities of  the OMIT map to fit that of  the initial map is negligible. This is perhaps not surprising a posteriori, since the application of histogram modification is important only for the real-space correlation-coefficient calculation and not for the map quality, since it is simply a nonlinear rescaling of the map and influences only the choice of  the isoline level. (iv) The most surprising result has been that, in the regions of  the map where the model is incorrect, with a low real-space correlation coefficient, very interesting OMIT maps (minimum correlation 0.416) are obtained when only the Sim figure-of- merit is used as the initial map amplitude coefficient (Fig. 1 c). This indicates, once again, that phases are more important than structure-factor amplitudes when it comes to the calculation of an electron-density distribution. Thus, these tests suggest that this type of  OMIT map, together with OMIT maps computed with IFol and with modified SIGMAA coefficients as the initial amplitude, are particularly useful to reconstruct the macro- molecular model in the grossly incorrect regions of  the model. It has not escaped our attention that such OMIT maps may also be used as the starting point for electron-density modification procedures. Tests are currently being carried out using the DEMON~ANGEL density-modification software suite (Vellieux, Hunt, Roy & Read, 1995) to investigate such OMIT maps in this context. It can also be suggested, from the results summarized in Table 1, that the use of  several OMIT maps, computed with different initial amplitude coefficients (i.e. only the Sim figure of  merit m, IFol and modified SIGMAA coefficients of  the form 3m IFol - 2DIFcI for acentric reflections and 2mlFol - DIFcI for centric reflections) may be an optimal strategy in the detection of  errors in a macromolecular model. The OMIT program is available freely upon request to the authors (e-mail vellieux@ibs.ibs.fr or bauke@chem.rug.nl). S H O R T  C O M M U N I C A T I O N S  399 The members of the lnstitut de Biologie Structurale Jean- Pierre Ebel CEA CNRS dedicate this paper to the memory of Rosaria and Serge Pares, who died on board TWA flight 800 off the Long Island coast. We thank Professor A. T. Briinger (Yale University) for having provided the prerelease of X-PLOR Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading of  the manuscript. Financial support of the French CEA and CNRS, and of the Netherlands Foundation for Chemical Research (SON), is gratefully acknowledged. References Bhat, T. N & Cohen, G. H (1984). J Appl. Cryst. 17, 244-248. Bhat, T. N. (1988). J Appl. Cryst. 21,279-281. Briinger, A. T., Kuriyan, J. & Karplus, M. (1987). Science, 235, 458-460. Hodel, A., Kim, S.-H. & Briinger, A. T. (1992). Acta Cryst. A48, 85 i-859. Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110-119. Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst. D52, 826-828. Lunin, V. Y. (1988). Acta Cryst. A44, 144-150. Read, R. J. (1986). Acta Cryst. A42, 140-149. Sim, G. A. (1959). Acta Cryst. 12, 813-815. Sim, G. A. (1960). Acta Cryst. 13, 511-512. Vellieux, E M. D. A. P., Hunt, J. F., Roy, S. & Read, R. J. (1995). J. Appl. Cryst. 28, 347-351. Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, J. C. (1997). In preparation. Vijayan, M. (1980). Acta Cryst. A36, 295-298. Zhang, K. Y. J. & Main, P. (1990). Acta Cryst. A46, 41-46. 

University of Groningen Research Database

  
 University of Groningen
Computation of Bhat's OMIT maps with different coefficients
Vellieux, FMD; Dijkstra, BW
Published in:
Journal of Applied Crystallography
DOI:
10.1107/S0021889896012551
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1997
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Vellieux, F. M. D., & Dijkstra, B. W. (1997). Computation of Bhat's OMIT maps with different coefficients.
Journal of Applied Crystallography, 30(3), 396-399. DOI: 10.1107/S0021889896012551
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
396 SHORT COMMUNICATIONS 
J Appl. Cryst. (1997). 30, 396-399 
Computat ion  o f  Bhat's OMIT  maps  with dif ferent coeff icients 
E M. D. VELLIEUX a AND B. W. DIJKSTRA b at %aboratoire de Cristallographie et de CristallogenOse des Prot~ines, Institut de 
Biologie Structurale J an-Pierre Ebel CEA CNRS, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 01, France, and bBIOSON 
Research Institute and Laboratory of Biophysical Chemistry, State University of Groningen, Nijenborgh 4, 9747 AG Groningen, 
The Netherlands. E-mail: vellieux@ibs.ibs.fr 
(Received 5 August 1996; accepted 7 October 1996) 
Abstract 
The OMIT electron-density-map calculation of Bhat [Bhat & 
Cohen (1984)..I. Appl. Cryst. 17, 244-248; Bhat (1988). J. 
Appl. Cryst. 21,279-281] is very effective in discovering errors 
in a macromolecular structure determination. A Fortran 
program called OMIT has been written to calculate such maps 
and an investigation has been carried out into which coefficients 
for the map calculation produce the best OMIT maps. Testing of 
the program on Savinase ~ showed that the best overall results 
were obtained when ]Fol without figure of merit was used. In 
regions where the map is incorrect, the most interesting OMIT 
maps are produced when only the figure of merit, or modified 
SIGMAA coefficients, are used as the initial map amplitude 
coefficients. Thus, these tests suggest that such OMIT maps are 
particularly useful to reconstruct the macromolecular model in 
the grossly incorrect regions of the model. 
1. Introduction 
The OMIT electron-density maps computed using Bhat's 
procedure (Bhat & Cohen, 1984; Bhat, 1988) are known to 
be very effective for the discovery of errors in the crystal 
structure determination of macromolecules. However, in their 
publications, Bhat and coauthor have not investigated the 
effects of different Fourier coefficients on the quality of the 
resulting OMIT maps. The subject of this paper is the study of 
several types of amplitude coefficients for the computation of 
the initial electron-density distribution from which OMIT maps 
are calculated, and the analysis of the resulting maps. 
2. Definitions 
IFol and IFcl are the observed and calculated structure-factor 
amplitudes, respectively, and IEol is the normalized structure- 
factor amplitude for the observed reflection, m is Sim's figure of 
merit, m = 1 I(X)/Io(X ) for acentric reflections and 
m = tanh(X/2), where 11 and I 0 are the modified Bessel 
functions of order 1 and 0, respectively, and X = 2lEo liE c I/E L 
(~L = EF,~) 
SIGMAA: Read (1986) has shown that electron-density maps 
calculated with amplitude coefficients of the form 
2mlFol- DIF~I for acentric reflections and mlFol for centric 
reflections are less biased towards the macromolecular model 
from which the IFcl's are calculated. In this work, we have used 
modified SIGMAA coefficients of the form 3mlFol - 2Olfcl for 
acentric reflections and 2mlFol- DIF~I for centric reflections 
(vide infra). 
OMIT maps: the reader is referred to Bhat (1988) for a 
complete description of the reciprocal-space procedure used to 
compute such maps, in which the electron density of a 'phasing 
volume' plus that of a 'neutral volume' surrounding it do not 
contribute to the IFcl's and $c's used in the procedure. 
CCF 1 is a real-space correlation coefficient with respect o a 
'control map', without any resolution cut-off, computed from 
refined atomic coordinates. CCF2 is a correlation coefficient 
between the OMIT map and a 2.0A-resolution artificial 
electron-density distribution generated from refined atomic 
coordinates. 
3. Material and methods 
A Fortran program called OMITwas written that allows one to 
compute Bhat's OMIT maps in a single step. This program 
requires amplitudes and phases in the CCP4 (Collaborative 
Computational Project, Number 4, 1994) MTZ format, and 
generates an OMIT map in the Groningen BIOMOL Master 
Fourier Map (MFF) format, which can be converted to other 
formats (e.g. DSN6, CCP4, X-PLOR) using separate programs 
that are available from the authors. 
In this study, we have used 'classical' coefficients of the form 
Eo, Fo, mFo, 2F o -F  c etc. (Table 1) to compute the initial 
electron-density distribution from which Bhat's OMIT maps are 
calculated. Concomitantly, we have developed a new type of 
computation of OMIT maps: in the first step, a 'classical' 
electron-density map is computed and the histogram of 
densities (Lunin, 1988; Zhang & Main, 1990) of this map is 
generated. The OMIT map is then computed and, finally, the 
histogram of densities of the OMIT map is adjusted to fit that of 
the initial electron-density map. 
All our tests have been carried out using models of the 
structure of monoclinic Savinase ~ in complex with the active 
site inhibitor benzo-furan boronic acid (BCsO3HT) (Verger, 
Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997). We used 
the 2.0 A-resolution unrefined model for the computation of the 
calculated amplitudes and phases used in the OMIT-map 
calculations. For the computation of the real-space correlation 
coefficient CCF1 between the observed OMIT density and the 
calculated ensity generated from the refined molecular model, 
carried out with the program. O (Jones, Zou, Cowan & 
Kjeldgaard, 1991), the 1.7A-resolution refined model of 
Savinase ~ was used to generate a calculated ensity with no 
resolution cut-off. We also computed, using the program 
MAPMAN (Kleywegt & Jones, 1996), the correlation coeffi- 
cient CCF2 between the OMIT maps and a 2.0 ,~-resolution 
artificial electron-density distribution generated from refined 
atomic coordinates. 
Savinase ~ crystallizes in space group P21, with cell 
dimensions a = 40.83, b = 72.91 and c- -  76.27 ,~, and ~ = 
90.0, fl = 91.21 amd ~/= 90.0 °. The crystals contain two 
molecules of Savinase @ in the asymmetric unit. For the OMIT- 
map calculations, the asymmetric unit was divided into 30 
phased volumes, each surrounded by a neutral volume 
© 1997 Intemational Union of Crystallography 
Printed in Great Britain - all fights reserved 
Journal of Applied Crystallography 
ISSN 0021-8898 © 1997 
SHORT COMMUNICATIONS 397 
,0068, 
(a) (b) (c) 
(d)  (e) 
Fig. 1. OMIT electron-density maps, computed with different initial amplitude coefficients. Contrary to the electron densities usually shown, this 
figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is 
evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density 
suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two 
conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. 
(a) 2.0 ~,-resolution OMIT map computed with initial amplitude coefficients of the form 3 [Fo[ - 2[Fc[ and phases from the unrefined model of 
Savinase @. (b) As (a), with initial amplitude coefficients of the form [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), 
with modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - D[Fc[ for the acentric reflections. (e) 1.7 ~,-resolution OMIT map 
computed with modified SIGMAA initial amplitude coefficients and phases from the refined model of Savinase @. This map w.as computed using 
the prerelease of X-PLORVersion 4.0 (Briinger, personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model 
of Savinase ~ before refinement; thick lines: 1.7 ,~-resolution model of Savinase ~ after efinement with X-PLOR. All maps were displayed using 
the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) and are contoured at the 1.0a level. 
398 SHORT COMMUNICAT IONS 
Table 1. Summary of  the results obtained with several amplitude coefficients for the computation of  the initial electron-density 
distribution from which the OMIT map is calculated 
HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), 
except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space 
correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- 
density distribution obtained from refined atomic oordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a 
residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation 
coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- 
resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients 
are shown in bold. 
With HM Without HM 
Coefficient Min CCF1 Max CCFI (CCFI) Min CCFI Max CCFI (CCF1) CCF2 
IEol 0.390 0.852 0.659 0.390 0.849 0.663 0.478 
IFol 0.380 0.856 0.663 0.396 0.853 0.669 0.479 
mlFol 0.348 0.844 0.643 0.348 0.844 0.644 0.454 
21Fol - IFcl 0.351 0.859 0.652 0.351 0.858 0.651 0.273 
m(2lFol- IFcl) 0.328 0.850 0.637 0.329 0.850 0.637 0.439 
3 IFol - 2lFcl 0.252 0.837 0.605 0.251 0.838 0.604 0.395 
m(31Fol - 21F¢1) 0.255 0.841 0.622 0.257 0.843 0.621 0.416 
3mlFol - 2DIFcl 0.349 0.862 0.644 0.332 0.864 0.638 0.449 
m 0.413 0.827 0.653 0.416 0.826 0.657 0.469 
consisting of two grid points in each direction (terms are 
defined as in Bhat, 1988). Since simulated-annealing OMIT- 
map calculations (Hodel, Kim & Briinger, 1992) took 
exceedingly long to compute (so long that such a computation 
was not completed after more than 24 h CPU time on our three- 
processor DEC ALPHA server), we did not investigate the 
effect of such refinement procedures together with different 
initial amplitude coefficients on the quality of the resulting 
OMIT maps. These OMIT maps, including the step of 
histogram apping, may now be computed using the prerelease 
of X-PLOR Version 4.0 (Briinger, personal communication; 
Hodel, Kim & Briinger, 1992; Briinger, Kuriyan & Karplus, 
1987). 
4. Results 
Our results (Table 1 and Fig. 1) can be summarized as follows: 
(i) Resetting the electron density values less than -1.0 times 
the root-mean-square ( .m.s.) electron density to -1 .0 times the 
r.m.s, electron density, as suggested by Bhat (1988), gave 
poorer OMIT maps than those computed with omission of this 
step (data not shown). Hence, in all our tests, this tnmcation 
step was omitted, so that Ifml = Ifcl, Wf = 1.0 and W = W, 
for all reflections (these terms are defined as in Bhat, 1988). 
Attempts have not been made to vary the threshold or the 
resolution of the data to analyse the usefulness of non- 
negativity criteria in OMIT maps. 
(ii) The highest correlations between the OMIT map and the 
calculated maps from the refined molecular model are obtained 
when no figure-of-merit (Sim, 1959, 1960) scheme is used, 
except for the OMIT maps computed with initial 31/7o[ - 2[Fc[ 
amplitude coefficients and for modified SIGMAA initial 
amplitude coefficients of the form 3m[Fo[ - 2D[F~[ for acentric 
reflections and 2m[Fo[-D[F~[ for centric reflections (the 
modified SIGMAA coefficients give improved electron-density 
maps with respect o the 'classical' coefficients of the form 
2mlFol -  D[Fc[ for acentric reflections and m[Fol for centric 
reflections: because the crystallographic refinement problem is 
undetermined for protein structures, where we are usually far 
from atomic resolution, it is possible to overfit he amplitudes in 
structure refinement. The amplitude differences are therefore 
smaller than they would be if the errors in the structure were 
truly random. To correct for that effect, in part, we can scale up 
the contribution of the differences in the electron-density-map 
coefficients (Vellieux, unpublished work; Read, 1986; Vijayan, 
1980). 
(iii) The effect of adjusting the histogram of densities of the 
OMIT map to fit that of the initial map is negligible. This is 
perhaps not surprising a posteriori, since the application of 
histogram modification is important only for the real-space 
correlation-coefficient calculation and not for the map quality, 
since it is simply a nonlinear rescaling of the map and 
influences only the choice of the isoline level. 
(iv) The most surprising result has been that, in the regions of 
the map where the model is incorrect, with a low real-space 
correlation coefficient, very interesting OMIT maps (minimum 
correlation 0.416) are obtained when only the Sim figure-of- 
merit is used as the initial map amplitude coefficient (Fig. 1 c). 
This indicates, once again, that phases are more important than 
structure-factor amplitudes when it comes to the calculation of 
an electron-density distribution. Thus, these tests suggest hat 
this type of OMIT map, together with OMIT maps computed 
with IFol and with modified SIGMAA coefficients as the initial 
amplitude, are particularly useful to reconstruct the macro- 
molecular model in the grossly incorrect regions of the model. 
It has not escaped our attention that such OMIT maps may 
also be used as the starting point for electron-density 
modification procedures. Tests are currently being carried out 
using the DEMON~ANGEL density-modification software suite 
(Vellieux, Hunt, Roy & Read, 1995) to investigate such OMIT 
maps in this context. It can also be suggested, from the results 
summarized in Table 1, that the use of several OMIT maps, 
computed with different initial amplitude coefficients (i.e. only 
the Sim figure of merit m, IFol and modified SIGMAA 
coefficients of the form 3m IFol - 2DIFcI for acentric reflections 
and 2mlFol - DIFcI for centric reflections) may be an optimal 
strategy in the detection of errors in a macromolecular model. 
The OMIT program is available freely upon request o the 
authors (e-mail vellieux@ibs.ibs.fr o bauke@chem.rug.nl). 
SHORT COMMUNICAT IONS 399 
The members of the lnstitut de Biologie Structurale Jean- 
Pierre Ebel CEA CNRS dedicate this paper to the memory of 
Rosaria and Serge Pares, who died on board TWA flight 800 off 
the Long Island coast. We thank Professor A. T. Briinger (Yale 
University) for having provided the prerelease of X-PLOR 
Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading 
of the manuscript. Financial support of the French CEA and 
CNRS, and of the Netherlands Foundation for Chemical 
Research (SON), is gratefully acknowledged. 
References 
Bhat, T. N & Cohen, G. H (1984). J Appl. Cryst. 17, 244-248. 
Bhat, T. N. (1988). J Appl. Cryst. 21,279-281. 
Briinger, A. T., Kuriyan, J. & Karplus, M. (1987). Science, 235, 
458-460. 
Hodel, A., Kim, S.-H. & Briinger, A. T. (1992). Acta Cryst. A48, 
85 i-859. 
Collaborative Computational Project, Number 4 (1994). Acta Cryst. 
D50, 760-763. 
Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta 
Cryst. A47, 110-119. 
Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst. D52, 826-828. 
Lunin, V. Y. (1988). Acta Cryst. A44, 144-150. 
Read, R. J. (1986). Acta Cryst. A42, 140-149. 
Sim, G. A. (1959). Acta Cryst. 12, 813-815. 
Sim, G. A. (1960). Acta Cryst. 13, 511-512. 
Vellieux, E M. D. A. P., Hunt, J. F., Roy, S. & Read, R. J. (1995). J. 
Appl. Cryst. 28, 347-351. 
Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, 
J. C. (1997). In preparation. 
Vijayan, M. (1980). Acta Cryst. A36, 295-298. 
Zhang, K. Y. J. & Main, P. (1990). Acta Cryst. A46, 41-46. 


University of Groningen

   University of GroningenComputation of Bhat's OMIT maps with different coefficientsVellieux, FMD; Dijkstra, BWPublished in:Journal of Applied CrystallographyDOI:10.1107/S0021889896012551IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vellieux, FMD., & Dijkstra, BW. (1997). Computation of Bhat's OMIT maps with different coefficients.Journal of Applied Crystallography, 30(3), 396-399. https://doi.org/10.1107/S0021889896012551CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 27-04-2023396 SHORT C O M M U N I C A T I O N S  J Appl. Cryst. (1997). 30, 396-399 C o m p u t a t i o n  o f  Bhat ' s  O M I T  m a p s  wi th  d i f ferent  coeff ic ients  E M. D. VELLIEUX a AND B. W. DIJKSTRA b at %aboratoire de Cristallographie et de CristallogenOse des Prot~ines, Institut de Biologie Structurale Jean-Pierre Ebel CEA CNRS, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 01, France, and bBIOSON Research Institute and Laboratory of  Biophysical Chemistry, State University of  Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: vellieux@ibs.ibs.fr (Received 5 August 1996; accepted 7 October 1996) Abstract The OMIT electron-density-map calculation of Bhat [Bhat & Cohen (1984)..I. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21,279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase ~ showed that the best overall results were obtained when ]Fol without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model. 1. Introduction The OMIT electron-density maps computed using Bhat's procedure (Bhat & Cohen, 1984; Bhat, 1988) are known to be very effective for the discovery of errors in the crystal structure determination of macromolecules. However, in their publications, Bhat and coauthor have not investigated the effects of different Fourier coefficients on the quality of the resulting OMIT maps. The subject of this paper is the study of several types of amplitude coefficients for the computation of the initial electron-density distribution from which OMIT maps are calculated, and the analysis of the resulting maps. 2. Definitions IFol and IFcl are the observed and calculated structure-factor amplitudes, respectively, and IEol is the normalized structure- factor amplitude for the observed reflection, m is Sim's figure of merit, m = 1 I(X)/Io(X ) for acentric reflections and m = tanh(X/2), where 11 and I 0 are the modified Bessel functions of order 1 and 0, respectively, and X = 2lEo liE c I /E L (~L = EF,~) SIGMAA: Read (1986) has shown that electron-density maps calculated with amplitude coefficients of the form 2mlFol-  DIF~I for acentric reflections and mlFol for centric reflections are less biased towards the macromolecular model from which the IFcl's are calculated. In this work, we have used modified SIGMAA coefficients of the form 3mlFol - 2Olfcl for acentric reflections and 2mlFol-  DIF~I for centric reflections (vide infra). OMIT maps: the reader is referred to Bhat (1988) for a complete description of the reciprocal-space procedure used to compute such maps, in which the electron density of a 'phasing volume' plus that of a 'neutral volume' surrounding it do not contribute to the IFcl's and $c's used in the procedure. CCF 1 is a real-space correlation coefficient with respect to a 'control map', without any resolution cut-off, computed from refined atomic coordinates. CCF2 is a correlation coefficient between the OMIT map and a 2.0A-resolution artificial electron-density distribution generated from refined atomic coordinates. 3. Material and methods A Fortran program called OMITwas written that allows one to compute Bhat's OMIT maps in a single step. This program requires amplitudes and phases in the CCP4 (Collaborative Computational Project, Number 4, 1994) MTZ format, and generates an OMIT map in the Groningen BIOMOL Master Fourier Map (MFF) format, which can be converted to other formats (e.g. DSN6, CCP4, X-PLOR) using separate programs that are available from the authors. In this study, we have used 'classical' coefficients of the form Eo, Fo, mFo, 2F o - F  c etc. (Table 1) to compute the initial electron-density distribution from which Bhat's OMIT maps are calculated. Concomitantly, we have developed a new type of computation of OMIT maps: in the first step, a 'classical' electron-density map is computed and the histogram of densities (Lunin, 1988; Zhang & Main, 1990) of this map is generated. The OMIT map is then computed and, finally, the histogram of densities of the OMIT map is adjusted to fit that of the initial electron-density map. All our tests have been carried out using models of the structure of monoclinic Savinase ~ in complex with the active site inhibitor benzo-furan boronic acid (BCsO3HT) (Verger, Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997). We used the 2.0 A-resolution unrefined model for the computation of the calculated amplitudes and phases used in the OMIT-map calculations. For the computation of the real-space correlation coefficient CCF1 between the observed OMIT density and the calculated density generated from the refined molecular model, carried out with the program. O (Jones, Zou, Cowan & Kjeldgaard, 1991), the 1.7A-resolution refined model of Savinase ~ was used to generate a calculated density with no resolution cut-off. We also computed, using the program MAPMAN (Kleywegt & Jones, 1996), the correlation coeffi- cient CCF2 between the OMIT maps and a 2.0 ,~-resolution artificial electron-density distribution generated from refined atomic coordinates. Savinase ~ crystallizes in space group P21, with cell dimensions a = 40.83, b = 72.91 and c - -  76.27 ,~, and ~ = 90.0, fl = 91.21 amd ~/= 90.0 °. The crystals contain two molecules of Savinase @ in the asymmetric unit. For the OMIT- map calculations, the asymmetric unit was divided into 30 phased volumes, each surrounded by a neutral volume © 1997 Intemational Union of Crystallography Printed in Great Britain - all fights reserved Journal of Applied Crystallography ISSN 0021-8898 © 1997 SHORT COMMUNICATIONS 397 ,0068, (a) (b) (c) ( d )  (e) Fig. 1. OMIT electron-density maps, computed with different initial amplitude coefficients. Contrary to the electron densities usually shown, this figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. (a) 2.0 ~,-resolution OMIT map computed with initial amplitude coefficients of  the form 3 [Fo[ - 2[Fc[ and phases from the unrefined model of Savinase @. (b) As (a), with initial amplitude coefficients of the form [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), with modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - D[Fc[ for the acentric reflections. (e) 1.7 ~,-resolution OMIT map computed with modified SIGMAA initial amplitude coefficients and phases from the refined model of Savinase @. This map w.as computed using the prerelease of  X-PLORVers ion  4.0 (Briinger, personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model of Savinase ~ before refinement; thick lines: 1.7 ,~-resolution model of Savinase ~ after refinement with X-PLOR.  All maps were displayed using the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) and are contoured at the 1.0a level. 398 SHORT C O M M U N I C A T I O N S  Table 1. Summary o f  the results obtained with several amplitude coefficients for  the computation o f  the initial electron-density distribution from which the OMIT map is calculated HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- density distribution obtained from refined atomic coordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients are shown in bold. With HM Without HM Coefficient Min CCF1 Max CCFI (CCFI) Min CCFI Max CCFI (CCF1) CCF2 IEol 0.390 0.852 0.659 0.390 0.849 0.663 0.478 IFol 0.380 0.856 0.663 0.396 0.853 0.669 0.479 mlFol 0.348 0.844 0.643 0.348 0.844 0.644 0.454 21Fol - IFcl 0.351 0.859 0.652 0.351 0.858 0.651 0.273 m(2lFol- IFcl) 0.328 0.850 0.637 0.329 0.850 0.637 0.439 3 IFol - 2lFcl 0.252 0.837 0.605 0.251 0.838 0.604 0.395 m(31Fol - 21F¢1) 0.255 0.841 0.622 0.257 0.843 0.621 0.416 3mlFol - 2DIFcl 0.349 0.862 0.644 0.332 0.864 0.638 0.449 m 0.413 0.827 0.653 0.416 0.826 0.657 0.469 consisting of two grid points in each direction (terms are defined as in Bhat, 1988). Since simulated-annealing OMIT- map calculations (Hodel, Kim & Briinger, 1992) took exceedingly long to compute (so long that such a computation was not completed after more than 24 h CPU time on our three- processor DEC ALPHA server), we did not investigate the effect of  such refinement procedures together with different initial amplitude coefficients on the quality of  the resulting OMIT maps. These OMIT maps, including the step of  histogram mapping, may now be computed using the prerelease of  X-PLOR Version 4.0 (Briinger, personal communication; Hodel, Kim & Briinger, 1992; Briinger, Kuriyan & Karplus, 1987). 4. Results Our results (Table 1 and Fig. 1) can be summarized as follows: (i) Resetting the electron density values less than - 1 . 0  times the root-mean-square (r.m.s.) electron density to - 1 . 0  times the r.m.s, electron density, as suggested by Bhat (1988), gave poorer OMIT maps than those computed with omission of  this step (data not shown). Hence, in all our tests, this tnmcation step was omitted, so that Ifml = Ifcl, Wf = 1.0 and W = W, for all reflections (these terms are defined as in Bhat, 1988). Attempts have not been made to vary the threshold or the resolution of the data to analyse the usefulness of  non- negativity criteria in OMIT maps. (ii) The highest correlations between the OMIT map and the calculated maps from the refined molecular model are obtained when no figure-of-merit (Sim, 1959, 1960) scheme is used, except for the OMIT maps computed with initial 31/7o[ - 2[Fc[ amplitude coefficients and for modified SIGMAA initial amplitude coefficients of  the form 3m[Fo[ - 2D[F~[ for acentric reflections and 2m[Fo[-D[F~[ for centric reflections (the modified SIGMAA coefficients give improved electron-density maps with respect to the 'classical' coefficients of  the form 2mlFo l -  D[Fc[ for acentric reflections and m[Fol for centric reflections: because the crystallographic refinement problem is undetermined for protein structures, where we are usually far from atomic resolution, it is possible to overfit the amplitudes in structure refinement. The amplitude differences are therefore smaller than they would be if the errors in the structure were truly random. To correct for that effect, in part, we can scale up the contribution of  the differences in the electron-density-map coefficients (Vellieux, unpublished work; Read, 1986; Vijayan, 1980). (iii) The effect of  adjusting the histogram of densities of  the OMIT map to fit that of  the initial map is negligible. This is perhaps not surprising a posteriori, since the application of histogram modification is important only for the real-space correlation-coefficient calculation and not for the map quality, since it is simply a nonlinear rescaling of the map and influences only the choice of  the isoline level. (iv) The most surprising result has been that, in the regions of  the map where the model is incorrect, with a low real-space correlation coefficient, very interesting OMIT maps (minimum correlation 0.416) are obtained when only the Sim figure-of- merit is used as the initial map amplitude coefficient (Fig. 1 c). This indicates, once again, that phases are more important than structure-factor amplitudes when it comes to the calculation of an electron-density distribution. Thus, these tests suggest that this type of  OMIT map, together with OMIT maps computed with IFol and with modified SIGMAA coefficients as the initial amplitude, are particularly useful to reconstruct the macro- molecular model in the grossly incorrect regions of  the model. It has not escaped our attention that such OMIT maps may also be used as the starting point for electron-density modification procedures. Tests are currently being carried out using the DEMON~ANGEL density-modification software suite (Vellieux, Hunt, Roy & Read, 1995) to investigate such OMIT maps in this context. It can also be suggested, from the results summarized in Table 1, that the use of  several OMIT maps, computed with different initial amplitude coefficients (i.e. only the Sim figure of  merit m, IFol and modified SIGMAA coefficients of  the form 3m IFol - 2DIFcI for acentric reflections and 2mlFol - DIFcI for centric reflections) may be an optimal strategy in the detection of  errors in a macromolecular model. The OMIT program is available freely upon request to the authors (e-mail vellieux@ibs.ibs.fr or bauke@chem.rug.nl). S H O R T  C O M M U N I C A T I O N S  399 The members of the lnstitut de Biologie Structurale Jean- Pierre Ebel CEA CNRS dedicate this paper to the memory of Rosaria and Serge Pares, who died on board TWA flight 800 off the Long Island coast. We thank Professor A. T. Briinger (Yale University) for having provided the prerelease of X-PLOR Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading of  the manuscript. Financial support of the French CEA and CNRS, and of the Netherlands Foundation for Chemical Research (SON), is gratefully acknowledged. References Bhat, T. N & Cohen, G. H (1984). J Appl. Cryst. 17, 244-248. Bhat, T. N. (1988). J Appl. Cryst. 21,279-281. Briinger, A. T., Kuriyan, J. & Karplus, M. (1987). Science, 235, 458-460. Hodel, A., Kim, S.-H. & Briinger, A. T. (1992). Acta Cryst. A48, 85 i-859. Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110-119. Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst. D52, 826-828. Lunin, V. Y. (1988). Acta Cryst. A44, 144-150. Read, R. J. (1986). Acta Cryst. A42, 140-149. Sim, G. A. (1959). Acta Cryst. 12, 813-815. Sim, G. A. (1960). Acta Cryst. 13, 511-512. Vellieux, E M. D. A. P., Hunt, J. F., Roy, S. & Read, R. J. (1995). J. Appl. Cryst. 28, 347-351. Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, J. C. (1997). In preparation. Vijayan, M. (1980). Acta Cryst. A36, 295-298. Zhang, K. Y. J. & Main, P. (1990). Acta Cryst. A46, 41-46. 

The OMIT electron-density-map calculation of Bhat [Bhat & Cohen (1984). J. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21, 279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase(TM) showed that the best overall results were obtained when \F-o\ without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model

NARCIS 

Proceedings - University of Groningen

   University of GroningenComputation of Bhat's OMIT maps with different coefficientsVellieux, FMD; Dijkstra, BWPublished in:Journal of Applied CrystallographyDOI:10.1107/S0021889896012551IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vellieux, FMD., & Dijkstra, BW. (1997). Computation of Bhat's OMIT maps with different coefficients.Journal of Applied Crystallography, 30(3), 396-399. https://doi.org/10.1107/S0021889896012551CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022396 SHORT C O M M U N I C A T I O N S  J Appl. Cryst. (1997). 30, 396-399 C o m p u t a t i o n  o f  Bhat ' s  O M I T  m a p s  wi th  d i f ferent  coeff ic ients  E M. D. VELLIEUX a AND B. W. DIJKSTRA b at %aboratoire de Cristallographie et de CristallogenOse des Prot~ines, Institut de Biologie Structurale Jean-Pierre Ebel CEA CNRS, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 01, France, and bBIOSON Research Institute and Laboratory of  Biophysical Chemistry, State University of  Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: vellieux@ibs.ibs.fr (Received 5 August 1996; accepted 7 October 1996) Abstract The OMIT electron-density-map calculation of Bhat [Bhat & Cohen (1984)..I. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21,279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase ~ showed that the best overall results were obtained when ]Fol without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model. 1. Introduction The OMIT electron-density maps computed using Bhat's procedure (Bhat & Cohen, 1984; Bhat, 1988) are known to be very effective for the discovery of errors in the crystal structure determination of macromolecules. However, in their publications, Bhat and coauthor have not investigated the effects of different Fourier coefficients on the quality of the resulting OMIT maps. The subject of this paper is the study of several types of amplitude coefficients for the computation of the initial electron-density distribution from which OMIT maps are calculated, and the analysis of the resulting maps. 2. Definitions IFol and IFcl are the observed and calculated structure-factor amplitudes, respectively, and IEol is the normalized structure- factor amplitude for the observed reflection, m is Sim's figure of merit, m = 1 I(X)/Io(X ) for acentric reflections and m = tanh(X/2), where 11 and I 0 are the modified Bessel functions of order 1 and 0, respectively, and X = 2lEo liE c I /E L (~L = EF,~) SIGMAA: Read (1986) has shown that electron-density maps calculated with amplitude coefficients of the form 2mlFol-  DIF~I for acentric reflections and mlFol for centric reflections are less biased towards the macromolecular model from which the IFcl's are calculated. In this work, we have used modified SIGMAA coefficients of the form 3mlFol - 2Olfcl for acentric reflections and 2mlFol-  DIF~I for centric reflections (vide infra). OMIT maps: the reader is referred to Bhat (1988) for a complete description of the reciprocal-space procedure used to compute such maps, in which the electron density of a 'phasing volume' plus that of a 'neutral volume' surrounding it do not contribute to the IFcl's and $c's used in the procedure. CCF 1 is a real-space correlation coefficient with respect to a 'control map', without any resolution cut-off, computed from refined atomic coordinates. CCF2 is a correlation coefficient between the OMIT map and a 2.0A-resolution artificial electron-density distribution generated from refined atomic coordinates. 3. Material and methods A Fortran program called OMITwas written that allows one to compute Bhat's OMIT maps in a single step. This program requires amplitudes and phases in the CCP4 (Collaborative Computational Project, Number 4, 1994) MTZ format, and generates an OMIT map in the Groningen BIOMOL Master Fourier Map (MFF) format, which can be converted to other formats (e.g. DSN6, CCP4, X-PLOR) using separate programs that are available from the authors. In this study, we have used 'classical' coefficients of the form Eo, Fo, mFo, 2F o - F  c etc. (Table 1) to compute the initial electron-density distribution from which Bhat's OMIT maps are calculated. Concomitantly, we have developed a new type of computation of OMIT maps: in the first step, a 'classical' electron-density map is computed and the histogram of densities (Lunin, 1988; Zhang & Main, 1990) of this map is generated. The OMIT map is then computed and, finally, the histogram of densities of the OMIT map is adjusted to fit that of the initial electron-density map. All our tests have been carried out using models of the structure of monoclinic Savinase ~ in complex with the active site inhibitor benzo-furan boronic acid (BCsO3HT) (Verger, Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997). We used the 2.0 A-resolution unrefined model for the computation of the calculated amplitudes and phases used in the OMIT-map calculations. For the computation of the real-space correlation coefficient CCF1 between the observed OMIT density and the calculated density generated from the refined molecular model, carried out with the program. O (Jones, Zou, Cowan & Kjeldgaard, 1991), the 1.7A-resolution refined model of Savinase ~ was used to generate a calculated density with no resolution cut-off. We also computed, using the program MAPMAN (Kleywegt & Jones, 1996), the correlation coeffi- cient CCF2 between the OMIT maps and a 2.0 ,~-resolution artificial electron-density distribution generated from refined atomic coordinates. Savinase ~ crystallizes in space group P21, with cell dimensions a = 40.83, b = 72.91 and c - -  76.27 ,~, and ~ = 90.0, fl = 91.21 amd ~/= 90.0 °. The crystals contain two molecules of Savinase @ in the asymmetric unit. For the OMIT- map calculations, the asymmetric unit was divided into 30 phased volumes, each surrounded by a neutral volume © 1997 Intemational Union of Crystallography Printed in Great Britain - all fights reserved Journal of Applied Crystallography ISSN 0021-8898 © 1997 SHORT COMMUNICATIONS 397 ,0068, (a) (b) (c) ( d )  (e) Fig. 1. OMIT electron-density maps, computed with different initial amplitude coefficients. Contrary to the electron densities usually shown, this figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. (a) 2.0 ~,-resolution OMIT map computed with initial amplitude coefficients of  the form 3 [Fo[ - 2[Fc[ and phases from the unrefined model of Savinase @. (b) As (a), with initial amplitude coefficients of the form [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), with modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - D[Fc[ for the acentric reflections. (e) 1.7 ~,-resolution OMIT map computed with modified SIGMAA initial amplitude coefficients and phases from the refined model of Savinase @. This map w.as computed using the prerelease of  X-PLORVers ion  4.0 (Briinger, personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model of Savinase ~ before refinement; thick lines: 1.7 ,~-resolution model of Savinase ~ after refinement with X-PLOR.  All maps were displayed using the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) and are contoured at the 1.0a level. 398 SHORT C O M M U N I C A T I O N S  Table 1. Summary o f  the results obtained with several amplitude coefficients for  the computation o f  the initial electron-density distribution from which the OMIT map is calculated HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- density distribution obtained from refined atomic coordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients are shown in bold. With HM Without HM Coefficient Min CCF1 Max CCFI (CCFI) Min CCFI Max CCFI (CCF1) CCF2 IEol 0.390 0.852 0.659 0.390 0.849 0.663 0.478 IFol 0.380 0.856 0.663 0.396 0.853 0.669 0.479 mlFol 0.348 0.844 0.643 0.348 0.844 0.644 0.454 21Fol - IFcl 0.351 0.859 0.652 0.351 0.858 0.651 0.273 m(2lFol- IFcl) 0.328 0.850 0.637 0.329 0.850 0.637 0.439 3 IFol - 2lFcl 0.252 0.837 0.605 0.251 0.838 0.604 0.395 m(31Fol - 21F¢1) 0.255 0.841 0.622 0.257 0.843 0.621 0.416 3mlFol - 2DIFcl 0.349 0.862 0.644 0.332 0.864 0.638 0.449 m 0.413 0.827 0.653 0.416 0.826 0.657 0.469 consisting of two grid points in each direction (terms are defined as in Bhat, 1988). Since simulated-annealing OMIT- map calculations (Hodel, Kim & Briinger, 1992) took exceedingly long to compute (so long that such a computation was not completed after more than 24 h CPU time on our three- processor DEC ALPHA server), we did not investigate the effect of  such refinement procedures together with different initial amplitude coefficients on the quality of  the resulting OMIT maps. These OMIT maps, including the step of  histogram mapping, may now be computed using the prerelease of  X-PLOR Version 4.0 (Briinger, personal communication; Hodel, Kim & Briinger, 1992; Briinger, Kuriyan & Karplus, 1987). 4. Results Our results (Table 1 and Fig. 1) can be summarized as follows: (i) Resetting the electron density values less than - 1 . 0  times the root-mean-square (r.m.s.) electron density to - 1 . 0  times the r.m.s, electron density, as suggested by Bhat (1988), gave poorer OMIT maps than those computed with omission of  this step (data not shown). Hence, in all our tests, this tnmcation step was omitted, so that Ifml = Ifcl, Wf = 1.0 and W = W, for all reflections (these terms are defined as in Bhat, 1988). Attempts have not been made to vary the threshold or the resolution of the data to analyse the usefulness of  non- negativity criteria in OMIT maps. (ii) The highest correlations between the OMIT map and the calculated maps from the refined molecular model are obtained when no figure-of-merit (Sim, 1959, 1960) scheme is used, except for the OMIT maps computed with initial 31/7o[ - 2[Fc[ amplitude coefficients and for modified SIGMAA initial amplitude coefficients of  the form 3m[Fo[ - 2D[F~[ for acentric reflections and 2m[Fo[-D[F~[ for centric reflections (the modified SIGMAA coefficients give improved electron-density maps with respect to the 'classical' coefficients of  the form 2mlFo l -  D[Fc[ for acentric reflections and m[Fol for centric reflections: because the crystallographic refinement problem is undetermined for protein structures, where we are usually far from atomic resolution, it is possible to overfit the amplitudes in structure refinement. The amplitude differences are therefore smaller than they would be if the errors in the structure were truly random. To correct for that effect, in part, we can scale up the contribution of  the differences in the electron-density-map coefficients (Vellieux, unpublished work; Read, 1986; Vijayan, 1980). (iii) The effect of  adjusting the histogram of densities of  the OMIT map to fit that of  the initial map is negligible. This is perhaps not surprising a posteriori, since the application of histogram modification is important only for the real-space correlation-coefficient calculation and not for the map quality, since it is simply a nonlinear rescaling of the map and influences only the choice of  the isoline level. (iv) The most surprising result has been that, in the regions of  the map where the model is incorrect, with a low real-space correlation coefficient, very interesting OMIT maps (minimum correlation 0.416) are obtained when only the Sim figure-of- merit is used as the initial map amplitude coefficient (Fig. 1 c). This indicates, once again, that phases are more important than structure-factor amplitudes when it comes to the calculation of an electron-density distribution. Thus, these tests suggest that this type of  OMIT map, together with OMIT maps computed with IFol and with modified SIGMAA coefficients as the initial amplitude, are particularly useful to reconstruct the macro- molecular model in the grossly incorrect regions of  the model. It has not escaped our attention that such OMIT maps may also be used as the starting point for electron-density modification procedures. Tests are currently being carried out using the DEMON~ANGEL density-modification software suite (Vellieux, Hunt, Roy & Read, 1995) to investigate such OMIT maps in this context. It can also be suggested, from the results summarized in Table 1, that the use of  several OMIT maps, computed with different initial amplitude coefficients (i.e. only the Sim figure of  merit m, IFol and modified SIGMAA coefficients of  the form 3m IFol - 2DIFcI for acentric reflections and 2mlFol - DIFcI for centric reflections) may be an optimal strategy in the detection of  errors in a macromolecular model. The OMIT program is available freely upon request to the authors (e-mail vellieux@ibs.ibs.fr or bauke@chem.rug.nl). S H O R T  C O M M U N I C A T I O N S  399 The members of the lnstitut de Biologie Structurale Jean- Pierre Ebel CEA CNRS dedicate this paper to the memory of Rosaria and Serge Pares, who died on board TWA flight 800 off the Long Island coast. We thank Professor A. T. Briinger (Yale University) for having provided the prerelease of X-PLOR Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading of  the manuscript. Financial support of the French CEA and CNRS, and of the Netherlands Foundation for Chemical Research (SON), is gratefully acknowledged. References Bhat, T. N & Cohen, G. H (1984). J Appl. Cryst. 17, 244-248. Bhat, T. N. (1988). J Appl. Cryst. 21,279-281. Briinger, A. T., Kuriyan, J. & Karplus, M. (1987). Science, 235, 458-460. Hodel, A., Kim, S.-H. & Briinger, A. T. (1992). Acta Cryst. A48, 85 i-859. Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110-119. Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst. D52, 826-828. Lunin, V. Y. (1988). Acta Cryst. A44, 144-150. Read, R. J. (1986). Acta Cryst. A42, 140-149. Sim, G. A. (1959). Acta Cryst. 12, 813-815. Sim, G. A. (1960). Acta Cryst. 13, 511-512. Vellieux, E M. D. A. P., Hunt, J. F., Roy, S. & Read, R. J. (1995). J. Appl. Cryst. 28, 347-351. Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, J. C. (1997). In preparation. Vijayan, M. (1980). Acta Cryst. A36, 295-298. Zhang, K. Y. J. & Main, P. (1990). Acta Cryst. A46, 41-46. 

   University of GroningenComputation of Bhat's OMIT maps with different coefficientsVellieux, FMD; Dijkstra, BWPublished in:Journal of Applied CrystallographyDOI:10.1107/S0021889896012551IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vellieux, FMD., & Dijkstra, BW. (1997). Computation of Bhat's OMIT maps with different coefficients.Journal of Applied Crystallography, 30(3), 396-399. https://doi.org/10.1107/S0021889896012551CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019396 SHORT COMMUNICATIONS J Appl. Cryst. (1997). 30, 396-399 Computat ion  o f  Bhat's OMIT  maps  with dif ferent coeff icients E M. D. VELLIEUX a AND B. W. DIJKSTRA b at %aboratoire de Cristallographie et de CristallogenOse des Prot~ines, Institut de Biologie Structurale J an-Pierre Ebel CEA CNRS, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 01, France, and bBIOSON Research Institute and Laboratory of Biophysical Chemistry, State University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: vellieux@ibs.ibs.fr (Received 5 August 1996; accepted 7 October 1996) Abstract The OMIT electron-density-map calculation of Bhat [Bhat & Cohen (1984)..I. Appl. Cryst. 17, 244-248; Bhat (1988). J. Appl. Cryst. 21,279-281] is very effective in discovering errors in a macromolecular structure determination. A Fortran program called OMIT has been written to calculate such maps and an investigation has been carried out into which coefficients for the map calculation produce the best OMIT maps. Testing of the program on Savinase ~ showed that the best overall results were obtained when ]Fol without figure of merit was used. In regions where the map is incorrect, the most interesting OMIT maps are produced when only the figure of merit, or modified SIGMAA coefficients, are used as the initial map amplitude coefficients. Thus, these tests suggest that such OMIT maps are particularly useful to reconstruct the macromolecular model in the grossly incorrect regions of the model. 1. Introduction The OMIT electron-density maps computed using Bhat's procedure (Bhat & Cohen, 1984; Bhat, 1988) are known to be very effective for the discovery of errors in the crystal structure determination of macromolecules. However, in their publications, Bhat and coauthor have not investigated the effects of different Fourier coefficients on the quality of the resulting OMIT maps. The subject of this paper is the study of several types of amplitude coefficients for the computation of the initial electron-density distribution from which OMIT maps are calculated, and the analysis of the resulting maps. 2. Definitions IFol and IFcl are the observed and calculated structure-factor amplitudes, respectively, and IEol is the normalized structure- factor amplitude for the observed reflection, m is Sim's figure of merit, m = 1 I(X)/Io(X ) for acentric reflections and m = tanh(X/2), where 11 and I 0 are the modified Bessel functions of order 1 and 0, respectively, and X = 2lEo liE c I/E L (~L = EF,~) SIGMAA: Read (1986) has shown that electron-density maps calculated with amplitude coefficients of the form 2mlFol- DIF~I for acentric reflections and mlFol for centric reflections are less biased towards the macromolecular model from which the IFcl's are calculated. In this work, we have used modified SIGMAA coefficients of the form 3mlFol - 2Olfcl for acentric reflections and 2mlFol- DIF~I for centric reflections (vide infra). OMIT maps: the reader is referred to Bhat (1988) for a complete description of the reciprocal-space procedure used to compute such maps, in which the electron density of a 'phasing volume' plus that of a 'neutral volume' surrounding it do not contribute to the IFcl's and $c's used in the procedure. CCF 1 is a real-space correlation coefficient with respect o a 'control map', without any resolution cut-off, computed from refined atomic coordinates. CCF2 is a correlation coefficient between the OMIT map and a 2.0A-resolution artificial electron-density distribution generated from refined atomic coordinates. 3. Material and methods A Fortran program called OMITwas written that allows one to compute Bhat's OMIT maps in a single step. This program requires amplitudes and phases in the CCP4 (Collaborative Computational Project, Number 4, 1994) MTZ format, and generates an OMIT map in the Groningen BIOMOL Master Fourier Map (MFF) format, which can be converted to other formats (e.g. DSN6, CCP4, X-PLOR) using separate programs that are available from the authors. In this study, we have used 'classical' coefficients of the form Eo, Fo, mFo, 2F o -F  c etc. (Table 1) to compute the initial electron-density distribution from which Bhat's OMIT maps are calculated. Concomitantly, we have developed a new type of computation of OMIT maps: in the first step, a 'classical' electron-density map is computed and the histogram of densities (Lunin, 1988; Zhang & Main, 1990) of this map is generated. The OMIT map is then computed and, finally, the histogram of densities of the OMIT map is adjusted to fit that of the initial electron-density map. All our tests have been carried out using models of the structure of monoclinic Savinase ~ in complex with the active site inhibitor benzo-furan boronic acid (BCsO3HT) (Verger, Vellieux., Kiersten Nielsen & Fontecilla-Camps, 1997). We used the 2.0 A-resolution unrefined model for the computation of the calculated amplitudes and phases used in the OMIT-map calculations. For the computation of the real-space correlation coefficient CCF1 between the observed OMIT density and the calculated ensity generated from the refined molecular model, carried out with the program. O (Jones, Zou, Cowan & Kjeldgaard, 1991), the 1.7A-resolution refined model of Savinase ~ was used to generate a calculated ensity with no resolution cut-off. We also computed, using the program MAPMAN (Kleywegt & Jones, 1996), the correlation coeffi- cient CCF2 between the OMIT maps and a 2.0 ,~-resolution artificial electron-density distribution generated from refined atomic coordinates. Savinase ~ crystallizes in space group P21, with cell dimensions a = 40.83, b = 72.91 and c- -  76.27 ,~, and ~ = 90.0, fl = 91.21 amd ~/= 90.0 °. The crystals contain two molecules of Savinase @ in the asymmetric unit. For the OMIT- map calculations, the asymmetric unit was divided into 30 phased volumes, each surrounded by a neutral volume © 1997 Intemational Union of Crystallography Printed in Great Britain - all fights reserved Journal of Applied Crystallography ISSN 0021-8898 © 1997 SHORT COMMUNICATIONS 397 ,0068, (a) (b) (c) (d)  (e) Fig. 1. OMIT electron-density maps, computed with different initial amplitude coefficients. Contrary to the electron densities usually shown, this figure displays the OMIT electron density for Arg143, which is the most difficult region of the map to interpret: at 2.0 A resolution, there is evidence for a double conformation for this side chain; at 1.7 ~, resolution, from new data collected from a new crystal, the OMIT electron density suggests the presence of only one conformer for this side chain. Note that refinement displaced the Arginine side chain from one of the two conformations to its correct single conformation, located midway between the two conformations observed at 2.0 A resolution for this side chain. (a) 2.0 ~,-resolution OMIT map computed with initial amplitude coefficients of the form 3 [Fo[ - 2[Fc[ and phases from the unrefined model of Savinase @. (b) As (a), with initial amplitude coefficients of the form [Fo]. (c) As (a), with initial amplitude coefficients of the form m. (d) As (a), with modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - D[Fc[ for the acentric reflections. (e) 1.7 ~,-resolution OMIT map computed with modified SIGMAA initial amplitude coefficients and phases from the refined model of Savinase @. This map w.as computed using the prerelease of X-PLORVersion 4.0 (Briinger, personal communication; Briinger, Kuriyan & Karplus, 1987). Thin lines: 2.0 A-resolution model of Savinase ~ before refinement; thick lines: 1.7 ,~-resolution model of Savinase ~ after efinement with X-PLOR. All maps were displayed using the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) and are contoured at the 1.0a level. 398 SHORT COMMUNICAT IONS Table 1. Summary of  the results obtained with several amplitude coefficients for the computation of  the initial electron-density distribution from which the OMIT map is calculated HM: mapping of the histogram of densities of the OMIT map to that of the initial map. m: figures of merit computed according to Sim (1959, 1960), except for the modified SIGMAA coefficients (m), where they are computed according to Read (1986). Min CCFI: minimum value of the real-space correlation coefficient, calculated with the program O (Jones, Zou, Cowan & Kjeldgaard, 1991) between the OMIT map and the artificial electron- density distribution obtained from refined atomic oordinates, without any resolution cut-off, for one of the molecules in the asymmetric unit on a residue-per-residue basis. Max CCFI: maximum value of the real-space correlation coefficient. (CCF1): average value of the real-space correlation coefficient. CCF2: correlation coefficient computed using the program MAPMAN (Kleywegt & Jones, 1996) between the OMIT map and the 2.0 ,~- resolution artificial electron-density distribution computed from the refined molecular model. The maximum values of the correlation coefficients are shown in bold. With HM Without HM Coefficient Min CCF1 Max CCFI (CCFI) Min CCFI Max CCFI (CCF1) CCF2 IEol 0.390 0.852 0.659 0.390 0.849 0.663 0.478 IFol 0.380 0.856 0.663 0.396 0.853 0.669 0.479 mlFol 0.348 0.844 0.643 0.348 0.844 0.644 0.454 21Fol - IFcl 0.351 0.859 0.652 0.351 0.858 0.651 0.273 m(2lFol- IFcl) 0.328 0.850 0.637 0.329 0.850 0.637 0.439 3 IFol - 2lFcl 0.252 0.837 0.605 0.251 0.838 0.604 0.395 m(31Fol - 21F¢1) 0.255 0.841 0.622 0.257 0.843 0.621 0.416 3mlFol - 2DIFcl 0.349 0.862 0.644 0.332 0.864 0.638 0.449 m 0.413 0.827 0.653 0.416 0.826 0.657 0.469 consisting of two grid points in each direction (terms are defined as in Bhat, 1988). Since simulated-annealing OMIT- map calculations (Hodel, Kim & Briinger, 1992) took exceedingly long to compute (so long that such a computation was not completed after more than 24 h CPU time on our three- processor DEC ALPHA server), we did not investigate the effect of such refinement procedures together with different initial amplitude coefficients on the quality of the resulting OMIT maps. These OMIT maps, including the step of histogram apping, may now be computed using the prerelease of X-PLOR Version 4.0 (Briinger, personal communication; Hodel, Kim & Briinger, 1992; Briinger, Kuriyan & Karplus, 1987). 4. Results Our results (Table 1 and Fig. 1) can be summarized as follows: (i) Resetting the electron density values less than -1.0 times the root-mean-square ( .m.s.) electron density to -1 .0 times the r.m.s, electron density, as suggested by Bhat (1988), gave poorer OMIT maps than those computed with omission of this step (data not shown). Hence, in all our tests, this tnmcation step was omitted, so that Ifml = Ifcl, Wf = 1.0 and W = W, for all reflections (these terms are defined as in Bhat, 1988). Attempts have not been made to vary the threshold or the resolution of the data to analyse the usefulness of non- negativity criteria in OMIT maps. (ii) The highest correlations between the OMIT map and the calculated maps from the refined molecular model are obtained when no figure-of-merit (Sim, 1959, 1960) scheme is used, except for the OMIT maps computed with initial 31/7o[ - 2[Fc[ amplitude coefficients and for modified SIGMAA initial amplitude coefficients of the form 3m[Fo[ - 2D[F~[ for acentric reflections and 2m[Fo[-D[F~[ for centric reflections (the modified SIGMAA coefficients give improved electron-density maps with respect o the 'classical' coefficients of the form 2mlFol -  D[Fc[ for acentric reflections and m[Fol for centric reflections: because the crystallographic refinement problem is undetermined for protein structures, where we are usually far from atomic resolution, it is possible to overfit he amplitudes in structure refinement. The amplitude differences are therefore smaller than they would be if the errors in the structure were truly random. To correct for that effect, in part, we can scale up the contribution of the differences in the electron-density-map coefficients (Vellieux, unpublished work; Read, 1986; Vijayan, 1980). (iii) The effect of adjusting the histogram of densities of the OMIT map to fit that of the initial map is negligible. This is perhaps not surprising a posteriori, since the application of histogram modification is important only for the real-space correlation-coefficient calculation and not for the map quality, since it is simply a nonlinear rescaling of the map and influences only the choice of the isoline level. (iv) The most surprising result has been that, in the regions of the map where the model is incorrect, with a low real-space correlation coefficient, very interesting OMIT maps (minimum correlation 0.416) are obtained when only the Sim figure-of- merit is used as the initial map amplitude coefficient (Fig. 1 c). This indicates, once again, that phases are more important than structure-factor amplitudes when it comes to the calculation of an electron-density distribution. Thus, these tests suggest hat this type of OMIT map, together with OMIT maps computed with IFol and with modified SIGMAA coefficients as the initial amplitude, are particularly useful to reconstruct the macro- molecular model in the grossly incorrect regions of the model. It has not escaped our attention that such OMIT maps may also be used as the starting point for electron-density modification procedures. Tests are currently being carried out using the DEMON~ANGEL density-modification software suite (Vellieux, Hunt, Roy & Read, 1995) to investigate such OMIT maps in this context. It can also be suggested, from the results summarized in Table 1, that the use of several OMIT maps, computed with different initial amplitude coefficients (i.e. only the Sim figure of merit m, IFol and modified SIGMAA coefficients of the form 3m IFol - 2DIFcI for acentric reflections and 2mlFol - DIFcI for centric reflections) may be an optimal strategy in the detection of errors in a macromolecular model. The OMIT program is available freely upon request o the authors (e-mail vellieux@ibs.ibs.fr o bauke@chem.rug.nl). SHORT COMMUNICAT IONS 399 The members of the lnstitut de Biologie Structurale Jean- Pierre Ebel CEA CNRS dedicate this paper to the memory of Rosaria and Serge Pares, who died on board TWA flight 800 off the Long Island coast. We thank Professor A. T. Briinger (Yale University) for having provided the prerelease of X-PLOR Version 4.0 and Dr M. Frey (IBS/LCCP) for his critical reading of the manuscript. Financial support of the French CEA and CNRS, and of the Netherlands Foundation for Chemical Research (SON), is gratefully acknowledged. References Bhat, T. N & Cohen, G. H (1984). J Appl. Cryst. 17, 244-248. Bhat, T. N. (1988). J Appl. Cryst. 21,279-281. Briinger, A. T., Kuriyan, J. & Karplus, M. (1987). Science, 235, 458-460. Hodel, A., Kim, S.-H. & Briinger, A. T. (1992). Acta Cryst. A48, 85 i-859. Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110-119. Kleywegt, G. J. & Jones, T. A. (1996). Acta Cryst. D52, 826-828. Lunin, V. Y. (1988). Acta Cryst. A44, 144-150. Read, R. J. (1986). Acta Cryst. A42, 140-149. Sim, G. A. (1959). Acta Cryst. 12, 813-815. Sim, G. A. (1960). Acta Cryst. 13, 511-512. Vellieux, E M. D. A. P., Hunt, J. F., Roy, S. & Read, R. J. (1995). J. Appl. Cryst. 28, 347-351. Verger, D., Vellieux, E M. D., Kiersten Nielsen, L. & Fontecilla-Camps, J. C. (1997). In preparation. Vijayan, M. (1980). Acta Cryst. A36, 295-298. Zhang, K. Y. J. & Main, P. (1990). Acta Cryst. A46, 41-46. 

Computation of Bhat's OMIT maps with different coefficients

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