As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of microdomains. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 8 m.Instituto de Investigaciones Bioquimicas de La Plat

Cooper, Alan

Córsico, Betina

Gabrielsen, Mads

Griffiths, Kate

Ibáñez Shimabukuro, Marina

Kennedy, Malcolm W.

Riboldi-Tunnicliffe, Alan

Roe, Andrew J.

Smith, Brian O.

Acta Crystallographica Section F Structural Biology and Crystallization Communications

Servicio de Difusión de la Creación Intelectual

crystallization communicationsActa Cryst. (2012). F68, 939–941 doi:10.1107/S1744309112026553 939Acta Crystallographica Section FStructural Biologyand CrystallizationCommunicationsISSN 1744-3091Useable diffraction data from a multiplemicrodomain-containing crystal of Ascaris suumAs-p18 fatty-acid-binding protein using amicrofocus beamlineMads Gabrielsen,a* Alan Riboldi-Tunnicliffe,b Marina Iba´n˜ez-Shimabukuro,c Kate Griffiths,dAndrew J. Roe,a Alan Cooper,eBrian O. Smith,f* Betina Co´rsicocand Malcolm W. Kennedyd,faInstitute of Infection, Immunity andInflammation, University of Glasgow, Glasgow,Scotland, bAustralian Synchrotron, Clayton,Victoria, Australia, cInstituto de InvestigacionesBioquimicas de La Plata, CONICET–UNLP,Facultad de Ciencias Medicas, UniversidadNacional de La Plata, La Plata, Argentina,dInstitute of Biodiversity, University of Glasgow,Glasgow, Scotland, eSchool of Chemistry,University of Glasgow, Glasgow, Scotland, andfInstitute of Molecular, Cell and SystemsBiology, University of Glasgow, Glasgow,ScotlandCorrespondence e-mail:mgabr@chem.gla.ac.uk,brian.smith@glasgow.ac.ukReceived 24 April 2012Accepted 12 June 2012As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum.Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystalswere obtained, but initial diffraction data analysis revealed that they werecomposed of a number of ‘microdomains’. Interpretable data could only becollected using a microfocus beamline with a beam size of 12  8 mm.1. IntroductionAs-p18 is a developmentally regulated fatty-acid-binding protein thatis one of the most abundant components of the perivitelline fluidsurrounding the developing larval stages of the parasitic nematodeAscaris suum (Mei et al., 1997). The infective third-stage (L3) larvaundergoes developmental arrest until ingestion by the host and maysurvive for up to seven years in the egg before infection of the host,but little is known of the biochemical and physiological basis ofsuch long-term survival. As-p18 displays sequence similarity tothe mammalian group of intracellular fatty-acid-binding proteins(iFABPs), but modelling indicated that there may be differences in itsligand-binding site and the existence of extended loops exposed onthe surface of the expected ten-stranded -barrel (Mei et al., 1997).These differences could relate to ligand specificity and interactionwith as yet uncharacterized protein or membrane interaction part-ners. Phylogenetic analysis reveals that As-p18, together with otherpotential homologues and paralogues in Caenorhabditis elegans,comprise a distinct protein class (‘nemFABPs’) within the FABPfamily that is unique to nematodes (Plenefisch et al., 2000). Thepresence of a leader peptide in mRNAs encoding As-p18 and directevidence of its secretion from the synthesizing cell further distinguishthis protein, and possibly nemFABPs in general, from FABPs foundin other animal groups. The atypical features of As-p18s may reflectadaptation to a specific function within the egg related to the survivalof nematode larvae in general, including those of the highly patho-genic agents of filariasis in humans (Kennedy & Harnett, 2001;Michalski et al., 2002).Here, we report crystallization conditions for As-p18 and the utilityof microfocus beamlines to analyse its structure. Whilst the crystalsshowed no obvious signs of disorder under optical microscopy, itbecame apparent from initial X-ray diffraction observations that thecrystals were composed of multiple microdomains. This preventeddata collection on a conventional beamline because the diffractionpatterns revealed the presence of multiple lattices. Only when thecrystals were brought to a microfocus beamline could a complete dataset be collected. The data-collection strategy and data processing ispresented here.2. Materials and methods2.1. Protein expression and purificationThe protein was expressed in Escherichia coli containing plasmidpQE-30/As-p18 (Mei et al., 1997), which encodes the protein with anN-terminal hexahistidine tag (His tag). The plasmid was sequence-verified and transformed into E. coli BL21 (DE3) cells harbouringplasmid pREP4, which encodes the lac repressor. Cells were grown inLB medium at 310 K, induced with 1.0 mM IPTG and harvested 4 hafter induction. The cells were lysed by sonication and the proteinwas purified from the supernatant by Ni–NTA (Novagen, Germany)affinity and size-exclusion (Superdex 75 HR 10/300; GE Healthcare,USA) chromatography.2.2. Crystallization and crystal preparationThe purified As-p18 with the His tag still intact was concentratedto approximately 15 mg ml1, as estimated by absorption at 280 nmusing a theoretical extinction coefficient of 28 420 M1 cm1 derivedfrom the aromatic amino-acid composition (Gasteiger et al., 2005).Crystallization trials were set up using commercially available screensand the sitting-drop vapour-diffusion method. The drops were set upwith a 1:1 ratio of protein and reservoir solution and a final volume of1 ml using a Cartesian nanodrop dispense unit (Digilab, USA). Thetrays were incubated at 293 K.Protein crystals appeared within two weeks in condition No. 2(40% ethylene glycol, 0.1 M acetate pH 4.5) of the Cryo I screen(Emerald BioSystems, USA). The largest crystals had maximumdimensions of 0.08  0.08  0.1 mm (Fig. 1). Crystals were cooledwithout any additional cryoprotection in a stream of gaseous nitrogencooled to 100 K (Oxford Cryosystems, England) and were storedunder liquid nitrogen until diffraction testing.3. Results3.1. Data collectionCrystals were initially tested at station I04 of Diamond LightSource (DLS), UK with an oscillation of 1 at a wavelength of0.9763 A˚. Although externally the crystals appeared to be wellformed, the diffraction spots were diffuse and showed signs ofaniosotropy (Fig. 2a). No indexing could be performed to assign thespace group.Using the (then) standard beam size of 71.8  75 mm at station I04,diffraction from several of these ‘microdomains’ was collected oneach image. A complete processable data set could therefore not becollected on a standard macromolecular crystallography beamline.Crystals from the same well were transported to station MX2 of theAustralian Synchrotron, where using the standard beam size of 35 20 mm resulted in similarly unusable diffraction patterns. In order toobtain useful data from these crystals, it was necessary to reduce thebeam size to 12  8 mm using the microcollimator installed on MX2.Further, owing to the presence of these microdomains within thecrystallization communications940 Gabrielsen et al.  As-p18 fatty-acid-binding protein Acta Cryst. (2012). F68, 939–941Figure 1Crystals of As-p18 obtained using 40% ethylene glycol, 0.1 M acetate pH 4.5.Figure 2X-ray diffraction images obtained (a) at station I04 of DLS using the standard beam size of 71.8  75 mm and (b) at station MX2 of the Australian Synchrotron using anoptimized beam size of 12  8 mm.crystal, it was necessary to screen several (20+) regions of the crystalusing the collimated beam.With a 10 mm aperture, it was possible to scan areas of the crystalto select an area that gave a single lattice in the diffraction pattern(Fig. 2b). A data set was collected from this region of the crystal, withdiffraction being observed to beyond 2.25 A˚ resolution. Data werecollected at a wavelength of 0.9537 A˚ on an ADSC Quantum 315rdetector (crystal-to-detector distance 380 mm) with an oscillation of0.5 using Blu-Ice (McPhillips et al., 2002).3.2. Data analysisThe crystal belonged to space group I4, with unit-cell parametersa = b = 102.254, c= 103.392 A˚. Owing to radiation damage, data couldbe processed to a maximum resolution of 2.3 A˚ using XDS (Kabsch,2010) and were reduced using SCALA (Evans, 2006). The data hadoverall Rmerge and hI/(I)i values of 8.4% and 6.8, respectively, andvalues of 49% and 1.4, respectively, in the highest resolution bin. Thespace group was confirmed by POINTLESS (Evans, 2006) and bymanual inspection of the systematic absences. A summary of thedata-processing statistics is presented in Table 1. With a molecularweight of 18 309 Da, the asymmetric unit is most likely to comprisethree copies of the protein with a solvent content of 53%. However,two copies and a solvent content of 69% is also possible. The correctcomposition has yet to be determined.4. ConclusionsWe successfully crystallized As-p18 and the crystals diffracted to aresolution of 2.3 A˚. Work is currently under way to solve the struc-ture using molecular-replacement methods based on the structures ofmammalian FABPs in the Protein Data Bank (PDB). The only waythat a quality diffraction pattern could be achieved was by using amicrofocus beamline, and this paper highlights the usefulness ofmicrofocus beamlines even for crystals that are apparently largeenough to be analysed using regular beamlines.This work was supported by Wellcome Trust grant No. 083625 toMWK, AC, BOS and BC, the National Research Council of Argen-tina (CONICET) fellowship to MIS and BC, and a Biotechnology andBiological Sciences Research Council grant to MG and AJR (BB/G011389/1). The expression plasmid for As-p18 was kindly providedby Rick Komuniecki, University of Toledo, Ohio, USA. Data werecollected on the MX2 beamline at the Australian Synchrotron,Victoria, Australia.ReferencesEvans, P. (2006). Acta Cryst. D62, 72–82.Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel,R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited byJ. M. Walker, pp. 571–607. Totowa: Humana Press.Kabsch, W. (2010). Acta Cryst. D66, 125–132.Kennedy, M. W. & Harnett, W. (2001). Parasitic Nematodes: MolecularBiology, Biochemistry and Immunology. New York, Wallingford: CABIPublishing.McPhillips, T. M., McPhillips, S. E., Chiu, H.-J., Cohen, A. E., Deacon, A. M.,Ellis, P. J., Garman, E., Gonzalez, A., Sauter, N. K., Phizackerley, R. P.,Soltis, S. M. & Kuhn, P. (2002). J. Synchrotron Rad. 9, 401–406.Mei, B., Kennedy, M. W., Beauchamp, J., Komuniecki, P. R. & Komuniecki, R.(1997). J. Biol. Chem. 272, 9933–9941.Michalski, M. L., Monsey, J. D., Cistola, D. P. & Weil, G. J. (2002). Mol.Biochem. Parasitol. 124, 1–10.Plenefisch, J., Xiao, H., Mei, B., Geng, J., Komuniecki, P. R. & Komuniecki, R.(2000). Mol. Biochem. Parasitol. 105, 223–236.crystallization communicationsActa Cryst. (2012). F68, 939–941 Gabrielsen et al.  As-p18 fatty-acid-binding protein 941Table 1Data-collection and reduction statistics.Values in parentheses are for the highest resolution bin.Space group I4Unit-cell parameters (A˚) a = 102.635, c = 103.392Resolution (A˚) 20.06–2.30 (2.42–2.30)Observed reflections 80135Unique reflections 21931Multiplicity 3.7 (1.9)Completeness (%) 93.2 (77.1)Matthews coefficient (A˚3 Da1) 3.97/2.65†Solvent content (%) 69.03/53.55†Monomers in asymmetric unit 2/3†Rmeas (%) 10.4 (67.9)Rp.i.m. (%) 4.9 (43.5)hI/(I)i 9.8 (1.8)Wilson B (A˚2) 41.7† The nature of the asymmetric unit has not yet been clarified, but it is expected tocontain two or three subunits.

Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline

SEDICI - Repositorio de la UNLP

crystallization communications
Acta Cryst. (2012). F68, 939–941 doi:10.1107/S1744309112026553 939
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Useable diffraction data from a multiple
microdomain-containing crystal of Ascaris suum
As-p18 fatty-acid-binding protein using a
microfocus beamline
Mads Gabrielsen,a* Alan Riboldi-
Tunnicliffe,b Marina Iba´n˜ez-
Shimabukuro,c Kate Griffiths,d
Andrew J. Roe,a Alan Cooper,e
Brian O. Smith,f* Betina Co´rsicoc
and Malcolm W. Kennedyd,f
aInstitute of Infection, Immunity and
Inflammation, University of Glasgow, Glasgow,
Scotland, bAustralian Synchrotron, Clayton,
Victoria, Australia, cInstituto de Investigaciones
Bioquimicas de La Plata, CONICET–UNLP,
Facultad de Ciencias Medicas, Universidad
Nacional de La Plata, La Plata, Argentina,
dInstitute of Biodiversity, University of Glasgow,
Glasgow, Scotland, eSchool of Chemistry,
University of Glasgow, Glasgow, Scotland, and
fInstitute of Molecular, Cell and Systems
Biology, University of Glasgow, Glasgow,
Scotland
Correspondence e-mail:
mgabr@chem.gla.ac.uk,
brian.smith@glasgow.ac.uk
Received 24 April 2012
Accepted 12 June 2012
As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum.
Although it exhibits sequence similarity to mammalian intracellular fatty-acid-
binding proteins, it contains features that are unique to nematodes. Crystals
were obtained, but initial diffraction data analysis revealed that they were
composed of a number of ‘microdomains’. Interpretable data could only be
collected using a microfocus beamline with a beam size of 12  8 mm.
1. Introduction
As-p18 is a developmentally regulated fatty-acid-binding protein that
is one of the most abundant components of the perivitelline fluid
surrounding the developing larval stages of the parasitic nematode
Ascaris suum (Mei et al., 1997). The infective third-stage (L3) larva
undergoes developmental arrest until ingestion by the host and may
survive for up to seven years in the egg before infection of the host,
but little is known of the biochemical and physiological basis of
such long-term survival. As-p18 displays sequence similarity to
the mammalian group of intracellular fatty-acid-binding proteins
(iFABPs), but modelling indicated that there may be differences in its
ligand-binding site and the existence of extended loops exposed on
the surface of the expected ten-stranded -barrel (Mei et al., 1997).
These differences could relate to ligand specificity and interaction
with as yet uncharacterized protein or membrane interaction part-
ners. Phylogenetic analysis reveals that As-p18, together with other
potential homologues and paralogues in Caenorhabditis elegans,
comprise a distinct protein class (‘nemFABPs’) within the FABP
family that is unique to nematodes (Plenefisch et al., 2000). The
presence of a leader peptide in mRNAs encoding As-p18 and direct
evidence of its secretion from the synthesizing cell further distinguish
this protein, and possibly nemFABPs in general, from FABPs found
in other animal groups. The atypical features of As-p18s may reflect
adaptation to a specific function within the egg related to the survival
of nematode larvae in general, including those of the highly patho-
genic agents of filariasis in humans (Kennedy & Harnett, 2001;
Michalski et al., 2002).
Here, we report crystallization conditions for As-p18 and the utility
of microfocus beamlines to analyse its structure. Whilst the crystals
showed no obvious signs of disorder under optical microscopy, it
became apparent from initial X-ray diffraction observations that the
crystals were composed of multiple microdomains. This prevented
data collection on a conventional beamline because the diffraction
patterns revealed the presence of multiple lattices. Only when the
crystals were brought to a microfocus beamline could a complete data
set be collected. The data-collection strategy and data processing is
presented here.
2. Materials and methods
2.1. Protein expression and purification
The protein was expressed in Escherichia coli containing plasmid
pQE-30/As-p18 (Mei et al., 1997), which encodes the protein with an
N-terminal hexahistidine tag (His tag). The plasmid was sequence-
verified and transformed into E. coli BL21 (DE3) cells harbouring
plasmid pREP4, which encodes the lac repressor. Cells were grown in
LB medium at 310 K, induced with 1.0 mM IPTG and harvested 4 h
after induction. The cells were lysed by sonication and the protein
was purified from the supernatant by Ni–NTA (Novagen, Germany)
affinity and size-exclusion (Superdex 75 HR 10/300; GE Healthcare,
USA) chromatography.
2.2. Crystallization and crystal preparation
The purified As-p18 with the His tag still intact was concentrated
to approximately 15 mg ml1, as estimated by absorption at 280 nm
using a theoretical extinction coefficient of 28 420 M1 cm1 derived
from the aromatic amino-acid composition (Gasteiger et al., 2005).
Crystallization trials were set up using commercially available screens
and the sitting-drop vapour-diffusion method. The drops were set up
with a 1:1 ratio of protein and reservoir solution and a final volume of
1 ml using a Cartesian nanodrop dispense unit (Digilab, USA). The
trays were incubated at 293 K.
Protein crystals appeared within two weeks in condition No. 2
(40% ethylene glycol, 0.1 M acetate pH 4.5) of the Cryo I screen
(Emerald BioSystems, USA). The largest crystals had maximum
dimensions of 0.08  0.08  0.1 mm (Fig. 1). Crystals were cooled
without any additional cryoprotection in a stream of gaseous nitrogen
cooled to 100 K (Oxford Cryosystems, England) and were stored
under liquid nitrogen until diffraction testing.
3. Results
3.1. Data collection
Crystals were initially tested at station I04 of Diamond Light
Source (DLS), UK with an oscillation of 1 at a wavelength of
0.9763 A˚. Although externally the crystals appeared to be well
formed, the diffraction spots were diffuse and showed signs of
aniosotropy (Fig. 2a). No indexing could be performed to assign the
space group.
Using the (then) standard beam size of 71.8  75 mm at station I04,
diffraction from several of these ‘microdomains’ was collected on
each image. A complete processable data set could therefore not be
collected on a standard macromolecular crystallography beamline.
Crystals from the same well were transported to station MX2 of the
Australian Synchrotron, where using the standard beam size of 35 
20 mm resulted in similarly unusable diffraction patterns. In order to
obtain useful data from these crystals, it was necessary to reduce the
beam size to 12  8 mm using the microcollimator installed on MX2.
Further, owing to the presence of these microdomains within the
crystallization communications
940 Gabrielsen et al.  As-p18 fatty-acid-binding protein Acta Cryst. (2012). F68, 939–941
Figure 1
Crystals of As-p18 obtained using 40% ethylene glycol, 0.1 M acetate pH 4.5.
Figure 2
X-ray diffraction images obtained (a) at station I04 of DLS using the standard beam size of 71.8  75 mm and (b) at station MX2 of the Australian Synchrotron using an
optimized beam size of 12  8 mm.
crystal, it was necessary to screen several (20+) regions of the crystal
using the collimated beam.
With a 10 mm aperture, it was possible to scan areas of the crystal
to select an area that gave a single lattice in the diffraction pattern
(Fig. 2b). A data set was collected from this region of the crystal, with
diffraction being observed to beyond 2.25 A˚ resolution. Data were
collected at a wavelength of 0.9537 A˚ on an ADSC Quantum 315r
detector (crystal-to-detector distance 380 mm) with an oscillation of
0.5 using Blu-Ice (McPhillips et al., 2002).
3.2. Data analysis
The crystal belonged to space group I4, with unit-cell parameters
a = b = 102.254, c= 103.392 A˚. Owing to radiation damage, data could
be processed to a maximum resolution of 2.3 A˚ using XDS (Kabsch,
2010) and were reduced using SCALA (Evans, 2006). The data had
overall Rmerge and hI/(I)i values of 8.4% and 6.8, respectively, and
values of 49% and 1.4, respectively, in the highest resolution bin. The
space group was confirmed by POINTLESS (Evans, 2006) and by
manual inspection of the systematic absences. A summary of the
data-processing statistics is presented in Table 1. With a molecular
weight of 18 309 Da, the asymmetric unit is most likely to comprise
three copies of the protein with a solvent content of 53%. However,
two copies and a solvent content of 69% is also possible. The correct
composition has yet to be determined.
4. Conclusions
We successfully crystallized As-p18 and the crystals diffracted to a
resolution of 2.3 A˚. Work is currently under way to solve the struc-
ture using molecular-replacement methods based on the structures of
mammalian FABPs in the Protein Data Bank (PDB). The only way
that a quality diffraction pattern could be achieved was by using a
microfocus beamline, and this paper highlights the usefulness of
microfocus beamlines even for crystals that are apparently large
enough to be analysed using regular beamlines.
This work was supported by Wellcome Trust grant No. 083625 to
MWK, AC, BOS and BC, the National Research Council of Argen-
tina (CONICET) fellowship to MIS and BC, and a Biotechnology and
Biological Sciences Research Council grant to MG and AJR (BB/
G011389/1). The expression plasmid for As-p18 was kindly provided
by Rick Komuniecki, University of Toledo, Ohio, USA. Data were
collected on the MX2 beamline at the Australian Synchrotron,
Victoria, Australia.
References
Evans, P. (2006). Acta Cryst. D62, 72–82.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel,
R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited by
J. M. Walker, pp. 571–607. Totowa: Humana Press.
Kabsch, W. (2010). Acta Cryst. D66, 125–132.
Kennedy, M. W. & Harnett, W. (2001). Parasitic Nematodes: Molecular
Biology, Biochemistry and Immunology. New York, Wallingford: CABI
Publishing.
McPhillips, T. M., McPhillips, S. E., Chiu, H.-J., Cohen, A. E., Deacon, A. M.,
Ellis, P. J., Garman, E., Gonzalez, A., Sauter, N. K., Phizackerley, R. P.,
Soltis, S. M. & Kuhn, P. (2002). J. Synchrotron Rad. 9, 401–406.
Mei, B., Kennedy, M. W., Beauchamp, J., Komuniecki, P. R. & Komuniecki, R.
(1997). J. Biol. Chem. 272, 9933–9941.
Michalski, M. L., Monsey, J. D., Cistola, D. P. & Weil, G. J. (2002). Mol.
Biochem. Parasitol. 124, 1–10.
Plenefisch, J., Xiao, H., Mei, B., Geng, J., Komuniecki, P. R. & Komuniecki, R.
(2000). Mol. Biochem. Parasitol. 105, 223–236.
crystallization communications
Acta Cryst. (2012). F68, 939–941 Gabrielsen et al.  As-p18 fatty-acid-binding protein 941
Table 1
Data-collection and reduction statistics.
Values in parentheses are for the highest resolution bin.
Space group I4
Unit-cell parameters (A˚) a = 102.635, c = 103.392
Resolution (A˚) 20.06–2.30 (2.42–2.30)
Observed reflections 80135
Unique reflections 21931
Multiplicity 3.7 (1.9)
Completeness (%) 93.2 (77.1)
Matthews coefficient (A˚3 Da1) 3.97/2.65†
Solvent content (%) 69.03/53.55†
Monomers in asymmetric unit 2/3†
Rmeas (%) 10.4 (67.9)
Rp.i.m. (%) 4.9 (43.5)
hI/(I)i 9.8 (1.8)
Wilson B (A˚2) 41.7
† The nature of the asymmetric unit has not yet been clarified, but it is expected to
contain two or three subunits.


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Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline

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