Respect the synchrotron beam strength: how to model it, measure it and mitigate it for various scientific fields

From Wiley via Jisc Publications RouterHistory: received 2021-07-26, accepted 2021-08-11, pub-electronic 2021-08-13Article version: VoRPublication status: PublishedExtremely bright synchrotron radiation sources give extremely strong intensities at the sample. Lawrence Bright et al (2021) [J. Synchrotron Rad. (2021), 28, https://doi.org/10.1107/S160057752100669X] dive into the details for materials science. I offer a Commentary including a historical context

How to Solve Protein Structures with an X-ray Laser

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The crystal structures of the enzyme hydroxymethylbilane synthase, also known as porphobilinogen deaminase

From Wiley via Jisc Publications RouterHistory: received 2021-08-10, accepted 2021-09-16, pub-print 2021-11-01, pub-electronic 2021-11-02Article version: VoRPublication status: PublishedThe enzyme hydroxymethylbilane synthase (HMBS; EC 4.3.1.8), also known as porphobilinogen deaminase, catalyses the stepwise addition of four molecules of porphobilinogen to form the linear tetrapyrrole 1‐hydroxymethylbilane. Thirty years of crystal structures are surveyed in this topical review. These crystal structures aim at the elucidation of the structural basis of the complex reaction mechanism involving the formation of tetrapyrrole from individual porphobilinogen units. The consistency between the various structures is assessed. This includes an evaluation of the precision of each molecular model and what was not modelled. A survey is also made of the crystallization conditions used in the context of the operational pH of the enzyme. The combination of 3D structural techniques, seeking accuracy, has also been a feature of this research effort. Thus, SAXS, NMR and computational molecular dynamics have also been applied. The general framework is also a considerable chemistry research effort to understand the function of the enzyme and its medical pathologies in acute intermittent porphyria (AIP). Mutational studies and their impact on the catalytic reaction provide insight into the basis of AIP and are also invaluable for guiding the understanding of the crystal structure results. Future directions for research on HMBS are described, including the need to determine the protonation states of key amino‐acid residues identified as being catalytically important. The question remains – what is the molecular engine for this complex reaction? Thermal fluctuations are the only suggestion thus far

Carboplatin binding to a model protein in non-NaCl conditions to eliminate partial conversion to cisplatin, and the use of different criteria to choose the resolution limit

Hen egg white lysozyme (HEWL) co-crystallisation conditions of carboplatin without sodium chloride (NaCl) have been utilised to eliminate partial conversion of carboplatin to cisplatin observed previously. Tetragonal HEWL crystals were successfully obtained in 65% MPD with 0.1M citric acid buffer at pH 4.0 including DMSO. The X-ray diffraction data resolution to be used for the model refinement was reviewed using several topical criteria together. The CC1/2 criterion implemented in XDS led to data being significant to 2.0{\AA}, compared to the data only being able to be processed to 3.0{\AA} using the Bruker software package (SAINT). Then using paired protein model refinements and DPI values based on the FreeR value, the resolution limit was fine tuned to be 2.3{\AA}. Interestingly this was compared with results from the EVAL software package which gave a resolution limit of 2.2{\AA} solely using crossing 2, but 2.8{\AA} based on the Rmerge values (60%). The structural results showed that carboplatin bound to only the N{\delta} binding site of His-15 one week after crystal growth, whereas five weeks after crystal growth, two molecules of carboplatin are bound to the His-15 residue. In summary several new results have emerged: - firstly non-NaCl conditions showed a carboplatin molecule bound to His-15 of HEWL; secondly binding of one molecule of carboplatin was seen after one week of crystal growth and two molecules were bound after five weeks of crystal growth; and thirdly the use of several criteria to determine the diffraction resolution limit led to the successful use of data to higher resolution.Comment: 14 pages; submitted to Acta Cryst D Biological Crystallography reference number tz504

Extensive counter-ion interactions seen at the surface of subtilisin in an aqueous medium

The extent of protein and counter-ion interactions in solution is still far from being fully described and understood. In low dielectric media there is documented evidence that counter-ions do bind and affect enzymatic activity. However, published crystal structures of macromolecules of biological interest in aqueous solution often do not report the presence of any counter-ions on the surface. The extent of counter-ion interactions within subtilisin in an aqueous medium has been investigated crystallographically using CsCl soak and X-ray wavelength optimised anomalous diffraction at the Cs K-edge. Ten Cs+, as well as six Cl- sites, have been clearly identified, revealing that in aqueous salt solutions ions can bind at defined points around the protein surface. The counter-ions do not generally interact with formal charges on the protein; formally neutral oxygens, mostly backbone carbonyls, mostly coordinate the Cs+ ions. The Cl- ion sites are also found likely to be near positive charges on the protein surface. The presence of counter-ions substantially changes the protein surface electrical charge. The surface charge distribution on a protein is commonly discussed in relation to enzyme function. The correct identification of counter-ions associated with a protein surface is necessary for a proper understanding of an enzyme's function

Crystallographic MAD Phasing Strategies Explored Using ELETTRA Sincrotrone Mn K-Edge Data to 2.1 Å and Use of CHESS Establishes the Diffraction Resolution Limit as 0.92 Å for the Protein Mn, Ca Concanavalin A

Multiwavelength anomalous dispersion (MAD) data have been collected from a single crystal of the protein concanavalin A so as to evaluate different combinations of wavelengths for crystallographic structure determination. Data were recorded to 2.1 Å resolution on a flash frozen crystal at three wavelengths about the Mn K-edge (1.8951 Å, 1.8940 Å, 1.800 Å) using synchrotron radiation at ELETTRA\u27s Sincrotrone Trieste \u27XRD\u27 beamline. This is one of the longest wavelength K-edge MAD studies undertaken to date. Anomalous and dispersive Patterson maps are seen to be of high quality and indicate a high occupancy for the manganese binding site. This is confirmed also in the MAD phase determination and electron density maps. Finally 0.92 Å data recorded at CHESS indicates the prospects available for combined phasing strategies based on MAD to medium/high resolution along with ultra high resolution data

Погляд на архіви

The International Union of Crystallography has for many years been advocating archiving of raw data to accompany structural papers. Recently, it initiated the formation of the Diffraction Data Deposition Working Group with the aim of developing standards for the representation of these data. A means of studying this issue is to submit exemplar publications with associated raw data and metadata. A recent study on the effects of dimethyl sulfoxide on the binding of cisplatin and carboplatin to histidine in 11 different lysozyme crystals from two diffractometers led to an investigation of the possible effects of the equipment and X-ray diffraction data processing software on the calculated occupancies and B factors of the bound Pt compounds. 35.3 Gb of data were transferred from Manchester to Utrecht to be processed with EVAL. A systematic comparison shows that the largest differences in the occupancies and B factors of the bound Pt compounds are due to the software, but the equipment also has a noticeable effect. A detailed description of and discussion on the availability of metadata is given. By making these raw diffraction data sets available via a local depository, it is possible for the diffraction community to make their own evaluation as they may wish

Cisplatin coordination chemistry determination at hen egg white lysozyme His15 with ligand distances and angles, and their standard uncertainties, and also reporting a split occupancy effect

Following the interest of L Messori and A Merlino 2016 Coordination Chemistry Reviews in the platinum ions coordination geometries in our PDB entries 4dd4 and 4dd6 we have extended our original analyses.Comment: 10 pages. One figure with two stereos and two further separate (identical to the stereos) figures with atom labels. This arXiv article is the first part of a Data Revie