Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase

Community recommendations on cryoEM data archiving and validation

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop’s motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.The workshop was supported by funding to PDBe and EMDB by the Wellcome Trust (grant No. 104948/Z/14/Z awarded to GJK, SV and AP) and by the European Molecular Biology Laboratory. Travel was supported by the PDBe, EMDB, RCSB PDB, PDBj, BMRB and EMDR. RCSB PDB is jointly funded by the National Science Foundation (grant No. DBI1832184); the US Department of Energy (grant No. DESC0019749); and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health (grant No. R01GM133198). PDBj is funded by JST-NBDC and BMRB by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) (grant No. R24GM150793). EMDR was funded by the NIGMS of the NIH (grant No. R01GM079429).Peer reviewe

The β-phase of Pigment Red 170: Structure Determination and Disorder Modelling

The single-crystal X-ray diffraction pattern from the β-phase of the industrially important Pigment Red 170 (β-P.R.170) consists of a difficult-to-disentangle mixture of Bragg diffraction superimposed by rods of diffuse scattering and satellite peaks. This extremely complicated diffraction pattern illustrates the complexity of real world crystals, whose underlying structure is far from the concept of a crystal being a regular periodic arrangement of unit cells usually presented in introductory crystallography textbooks. Such complex structures still present a big challenge to practitioners of X-ray crystallography. Understanding of the photochemical properties of this pigment would benefit from knowledge of the specific local arrangement of molecules in the crystal structure, but such information was not available due to the disordered nature of this material. The focus of this thesis was to model the crystal structure of this material by an analysis of the total diffraction pattern. The disorder in this material manifests itself as rods of strong diffuse scattering in the diffraction pattern. According to the mathematical description presented in the first part of the thesis, the type of disorder present in this material is stacking faults. These faults can occur during the stacking of the two dimensionally ordered molecular layers when the crystal grows. A detailed analysis of the diffraction pattern revealed that the rods of diffuse scattering pass through the Bragg reflections. Furthermore, it showed that a considerable percentage of the Bragg reflections is completely immersed in the strong diffuse streaks. As a result, the unit cell indexation and the accurate Bragg intensity estimation were extremely difficult. An analysis of only the Bragg reflections resulted in two plausible average structures. Both structures have the same unit cell dimensions, but occur in different space groups, namely B21/g and P21/a. The model developed in B21/g has only one symmetry-independent disordered molecular layer in which there are two symmetry-independent molecules, both of which are disordered over two positions with an occupancy ratio 0.91:0.09 related by the vector [0, -0.158b, 0]. In contrast, the model developed in P21/a has two symmetry independent molecular layers of which only one is disordered. The disordered layer is similar to the unique layer of the other model, but this time the occupancy ratio is 0.65:0.35. In addition, the two models differ in the number of molecules in the asymmetric unit, relative placement of molecular layers in the unit cell and the number of crystallographic and non-crystallographic symmetry elements in the average unit cell. The agreement R-factors calculated from both models implied that the B21/g model is the better description of the average structure. The basic structural unit in both models is the same. It possesses the layer group symmetry p 1 21/c 1. The geometries of all adjacent layer pairs in both models are equivalent. According to Order-Disorder theory, this implies that the two models belong to the same polytypic family, but they differ in their layer stacking sequences. The last part of the work presents the initial attempts taken to estimate the layer stacking sequence in the real crystal using model crystals. Two model crystals were constructed in the computer with the aid of a random number generator using the atomic coordinates and site occupancies obtained from the two average structures. The correlations between the interacting layers were introduced and the total interaction energy of each crystal was minimized according to the Monte Carlo (MC) method. The MC minimized crystals were then used to calculate total scattering intensities. Both disordered model crystals constructed and tested in this work produced broad diffuse scattering features superimposed with some fine structure. So far, the match with the experimental data is poor. It is not yet known whether the observed fine structure in each calculated pattern is due to some underlying periodicity of the molecular layers in the model crystal, or is just a consequence of the statistical noise in the MC simulations. Resolution of this problem will require future additional time-consuming calculations

In situ serial crystallography for rapid de novo membrane protein structure determination

De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Here we present a method that combines in situ serial crystallography with de novo phasing for fast, efficient membrane protein structure determination. The method enables systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without the need for direct crystal harvesting. The requisite data quality for experimental phasing is achieved by accumulating diffraction signals from isomorphous crystals identified post-data collection. The method works in all experimental phasing scenarios and is particularly attractive with fragile, weakly diffracting microcrystals. The automated serial data collection approach can be readily adopted at most microfocus macromolecular crystallography beamlines.publishe

TiGePt – a study of Friedel differences

Abstract: The X-ray single-crystal diffraction intensities of the intermetallic compound TiGePt were analysed. These showed beyond doubt that the crystal structure is non-centrosymmetric. The analysis revolves around the resonant-scattering contribution to differences in intensity between Friedel opposites hkl and \bar h\bar k\bar l. The following techniques were used: Rmerge factors on the average (A) and difference (D) of Friedel opposites; statistical estimates of the resonant-scattering contribution to Friedel opposites; plots of 2Aobs against 2Amodel and of Dobs against Dmodel; the antisymmetric D-Patterson function. Moreover it was possible to show that a non-standard atomic model was unnecessary to describe TiGePt. Two data sets are compared. That measured with Ag K[alpha] radiation at 295 K to a resolution of 1.25 Å-1 is less conclusive than the one measured with Mo K[alpha] radiation at 100 K to the lower resolution of 0.93 Å-1. This result is probably due to the fact that the resonant scattering of Pt is larger for Mo K[alpha] than for AgK[alpha] radiation

Pentaindenocorannulene: Properties, Assemblies, and C60 Complex

Pentaindenocorannulene (C50H20 , 1), a deep bowl polynuclear aromatic hydrocarbon, accepts 4 electrons, crystallizes in columnar bowl-in-bowl assemblies and forms a nested C60@12 complex. Spectra, structures and computations are presented

EIGER detector : application in macromolecular crystallography

The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed

In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshops motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations