Time-series analysis of rhenium(I) organometallic covalent binding to a model protein for drug development.

Metal-based complexes with their unique chemical properties, including multiple oxidation states, radio-nuclear capabilities and various coordination geometries yield value as potential pharmaceuticals. Understanding the interactions between metals and biological systems will prove key for site-specific coordination of new metal-based lead compounds. This study merges the concepts of target coordination with fragment-based drug methodologies, supported by varying the anomalous scattering of rhenium along with infrared spectroscopy, and has identified rhenium metal sites bound covalently with two amino acid types within the model protein. A time-based series of lysozyme-rhenium-imidazole (HEWL-Re-Imi) crystals was analysed systematically over a span of 38 weeks. The main rhenium covalent coordination is observed at His15, Asp101 and Asp119. Weak (i.e. noncovalent) interactions are observed at other aspartic, asparagine, proline, tyrosine and tryptophan side chains. Detailed bond distance comparisons, including precision estimates, are reported, utilizing the diffraction precision index supplemented with small-molecule data from the Cambridge Structural Database. Key findings include changes in the protein structure induced at the rhenium metal binding site, not observed in similar metal-free structures. The binding sites are typically found along the solvent-channel-accessible protein surface. The three primary covalent metal binding sites are consistent throughout the time series, whereas binding to neighbouring amino acid residues changes through the time series. Co-crystallization was used, consistently yielding crystals four days after setup. After crystal formation, soaking of the compound into the crystal over 38 weeks is continued and explains these structural adjustments. It is the covalent bond stability at the three sites, their proximity to the solvent channel and the movement of residues to accommodate the metal that are important, and may prove useful for future radiopharmaceutical development including target modification.

Raw diffraction data and reproducibility.

In recent years, there has been a major expansion in digital storage capability for hosting raw diffraction datasets. Naturally, the question has now arisen as to the benefits and costs for the preservation of such raw, i.e., experimental diffraction datasets. We describe the consultations made of the global structural chemistry, i.e., chemical crystallography community from the points of view of the International Union of Crystallography (IUCr) Committee on Data, of which JRH was the Chair until very recently, and the IUCrData Raw Data Letters initiative, for which LKB is the Main Editor. The monitoring by the CCDC of CSD depositions which cite the digital object identifiers of raw diffraction datasets provides interesting statistics by probe (x-ray, neutron, or electron) and by home lab vs central facility. Clearly, a better understanding of the reproducibility of current analysis procedures is at hand. Policies for publication requiring raw data have been updated in IUCr Journals for macromolecular crystallography, namely, that raw data should be made available for a new crystal structure or a new method as well as the wwPDB deposition. For chemical crystallography, such a step requiring raw data archiving has not yet been recommended by the IUCr Commission on Structural Chemistry.

The interoperability of crystallographic data and databases.

Interoperability of crystallographic data with other disciplines is essential for the smooth and rapid progress of structure-based science in the computer age. Within crystallography and closely related subject areas, there is already a high level of conformance to the generally accepted FAIR principles (that data be findable, accessible, interoperable and reusable) through the adoption of common information exchange protocols by databases, publishers, instrument vendors, experimental facilities and software authors. Driven by the success within these domains, the IUCr has worked closely with CODATA (the Committee on Data of the International Science Council) to help develop the latter's commitment to cross-domain integration of discipline-specific data. The IUCr has, in particular, emphasized the need for standards relating to data quality and completeness as an adjunct to the FAIR data landscape. This can ensure definitive reusable data, which in turn can aid interoperability across domains. A microsymposium at the IUCr 2023 Congress provided an up-to-date survey of data interoperability within and outside of crystallography, expounded using a broad range of examples.

Error estimates in atom coordinates and B factors in macromolecular crystallography.

The overall diffraction precision index (DPI) of a biological macromolecule crystal structure was first described by Cruickshank in 1999. This topical review proceeds from this point and describes the subsequent elaboration of the index to individual atom coordinates. Additional developments were introduced by the availability of a webserver, which provides a transformed PDB entry with individual atom coordinate errors derived from applying the DPI method using the parameters provided by the authors and then subsequently added to the PDB file. This webserver has been extensively used and harnessed in describing non-covalent distance error estimates as well as assessing the significance, or otherwise, of atom movements in a variety of studies. The standard uncertainties on a biological macromolecule's atomic displacement parameters (the 'B factors') has been an entirely different challenge but is obviously important since the crystallographic community has developed the habit of quoting B factors to a false precision in papers. This can convey a false certainty in the dynamics of a structure. A method involving parallelisation of workflows for diffraction image data processing does however offer estimates of the precision of B factors.

Observations on Laue diffraction within synchrotron radiation and neutron macromolecular crystallography research and developments.

A seminal contribution in the domain of physiologically relevant biological structure and function determination was by Keith Moffat, of Cornell and latterly of the University of Chicago proposing that synchrotrons should offer the option of a Laue method data collection mode. I enthusiastically joined in supporting this initiative. This proposal needed detailed methods development though; theoretical, experimental and software development. This work was added to the broad research and development program of synchrotron radiation at the UK's SRS. This whole program led to knowledge transfer from the UK's SRS to the ESRF as well as for neutron Laue protein crystallography to the reactor spallation sources and later to spallation neutron sources.

Golden oldies: ten crystallography articles that we think must be read.

We have selected a set of ten 'golden oldies', diverse crystallography articles to illustrate important moments in the development of our field of science and which form landmark papers in crystallography. They are a mixture of 'science pull and technology push'. For each of our choices, we firstly created a new title that emphasizes how the paper's importance worked out from today's perspective. Then we describe the core details and impacts of each paper, with some quotations and a selected figure or two. Ten is an arbitrary number of highlights and our choice is personal.

Relating protein crystal structure to ligand-binding thermodynamics.

An important interface between biophysical chemistry and biological crystal structures involves whether it is possible to relate experimental calorimetry measurements of protein ligand binding to 3D structures. This has proved to be challenging. The probes of the structure of matter, namely X-rays, neutrons and electrons, have challenges of one type or another in their use. This article focuses on saccharide binding to lectins as a theme, yet after 25 years or so it is still a work in progress to connect 3D structure to binding energies. Whilst this study involved one type of protein (lectins) and one class of ligand (monosaccharides), i.e. it was specific, it was of general importance, as measured for instance by its wide impact. The impetus for writing this update now, as a Scientific Comment, is that a breakthrough in neutron crystal structure determinations of saccharide-bound lectins has been achieved. It is suggested here that this new research from neutron protein crystallography could improve, i.e. reduce, the errors in the estimated binding energies.

The four Rs and crystal structure analysis: reliability, reproducibility, replicability and reusability.

Within science, of which crystallography is a key part, there are questions posed to all fields that challenge the trust in results. The US National Academies of Sciences, Engineering and Medicine published a thorough report in 2019 on the Reproducibility and Replicability of Science: replicability being where a totally new study attempts to confirm if a phenomenon can be seen independently of another study. Data reuse is a key term in the FAIR data accord [Wilkinson et al. (2016). Sci. Data, 3, 160018], where the acronym FAIR means findable, accessible, interoperable and reusable. In the social sciences, the acronym FACT (namely fairness, accuracy, confidentiality and transparency) has emerged, the idea being that data should be FACTual to ensure trust [van der Aalst et al. (2017). Bus. Inf. Syst. Eng. 59, 311-313]. A distinction also must be made between accuracy and precision; indeed, the authors' lectures at the European Crystallography School ECS6 independently emphasized the need for use of other methods as well as crystal structure analysis to establish accuracy in biological and chemical/material functional contexts. The efforts by disparate science communities to introduce new terms to ensure trust have merit for discussion in crystallographic teaching commissions and possible adoption by crystallographers too.

Raw diffraction data are our ground truth from which all subsequent workflows develop.

Defining best practice in science is challenging. International consensus is facilitated by the International Science Council via its members such as the International Union of Crystallography (IUCr). The crystallographic community has many decades of tradition linking articles with the underpinning data, and is admired across all sciences accordingly. Crystallography has always been at the forefront of harnessing new technology in the service of consensus. Technology has provided new vast data-archiving opportunities, allowing the preservation of raw diffraction data, along with article and database depositions of a model's coordinates and associated structure factors. The raw diffraction data, which can now be preserved, are the ground truth from which all subsequent workflows develop. Journal editorial boards provide a practical forum for setting the criteria to decide if a study's files are truly the version of record. Within that, reality involves a variance of reasonable workflows. But what is a reasonable variance? Workflows must be detailed carefully by authors in explaining what they have done. There is a great, and increasing, diversity of macromolecular crystallography analyses, and yet an increased constraint on how much can be written in an article about the workflow used. Raw data provide the ultimate reproducibility evidence. A part of reproducibility and replicability is using an agreed vocabulary; the meaning of words such as precision and accuracy and, more recently, the confidence of a protein structure prediction should feature in approaching `truth'.

Pre- and Post-publication Verification for Reproducible Data Mining in Macromolecular Crystallography.

Like an article narrative is deemed by an editor and referees to be worthy of being a version of record on acceptance as a publication, so must the underpinning data also be scrutinized before passing it as a version of record. Indeed without the underpinning data, a study and its conclusions cannot be reproduced at any stage of evaluation, pre- or post-publication. Likewise, an independent study without its own underpinning data also cannot be reproduced let alone be considered a replicate of the first study. The PDB is a modern marvel of achievement providing an organized open access to depositor and user of the data held there opening numerous applications. Methods for modeling protein structures and for determination of structures are still improving their precision, and artifacts of the method exist. So their accuracy is realized if they are reproduced by other methods. It is on such foundations that reproducible data mining is based. Data rates are expanding considerably be they at synchrotrons, the X-ray free electron lasers (XFELs), electron cryomicroscopes (cryoEM), or at the neutron facilities. The work of a person as a referee or user with a narrative and its underpinning data may well be complemented in future by artificial intelligence with machine learning, the former for specific refereeing and the latter for the more general validation, both ideally before publication. Examples are described involving rhenium theranostics, the anti-cancer platins and the SARS-CoV-2 main protease.

Trends in coordination of rhenium organometallic complexes in the Protein Data Bank.

Radiopharmaceutical development has similar overall characteristics to any biomedical drug development requiring a compound's stability, aqueous solubility and selectivity to a specific disease site. However, organometallic complexes containing 188/186Re or 99mTc involve a d-block transition-metal radioactive isotope and therefore bring additional factors such as metal oxidation states, isotope purity and half life into play. This topical review is focused on the development of radiopharmaceuticals containing the radioisotopes of rhenium and technetium and, therefore, on the occurrence of these organometallic complexes in protein structures in the Worldwide Protein Data Bank (wwPDB). The purpose of incorporating the group 7 transition metals of rhenium/technetium in the protein and the reasons for study by protein crystallography are described, as certain PDB studies were not aimed at drug development. Technetium is used as a medical diagnostic agent and involves the 99mTc isotope which decays to release gamma radiation, thereby employed for its use in gamma imaging. Due to the periodic relationship among group 7 transition metals, the coordination chemistry of rhenium is similar (but not identical) to that of technetium. The types of reactions the potential model radiopharmaceutical would prefer to partake in, and by extension knowing which proteins and biomolecules the compound would react with in vivo, are needed. Crystallography studies, both small molecule and macromolecular, are a key aspect in understanding chemical coordination. Analyses of bonding modes, coordination to particular residues and crystallization conditions are presented. In our Forward look as a concluding summary of this topical review, the question we ask is: what is the best way for this field to progress?

6-Phosphogluconate dehydrogenase and its crystal structures.

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyses the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate in the context of the oxidative part of the pentose phosphate pathway. Depending on the species, it can be a homodimer or a homotetramer. Oligomerization plays a functional role not only because the active site is at the interface between subunits but also due to the interlocking tail-modulating activity, similar to that of isocitrate dehydrogenase and malic enzyme, which catalyse a similar type of reaction. Since the pioneering crystal structure of sheep liver 6PGDH, which allowed motifs common to the ß-hydroxyacid dehydrogenase superfamily to be recognized, several other 6PGDH crystal structures have been solved, including those of ternary complexes. These showed that more than one conformation exists, as had been suggested for many years from enzyme studies in solution. It is inferred that an asymmetrical conformation with a rearrangement of one of the two subunits underlies the homotropic cooperativity. There has been particular interest in the presence or absence of sulfate during crystallization. This might be related to the fact that this ion, which is a competitive inhibitor that binds in the active site, can induce the same 6PGDH configuration as in the complexes with physiological ligands. Mutagenesis, inhibitors, kinetic and binding studies, post-translational modifications and research on the enzyme in cancer cells have been complementary to the crystallographic studies. Computational modelling and new structural studies will probably help to refine the understanding of the functioning of this enzyme, which represents a promising therapeutic target in immunity, cancer and infective diseases. 6PGDH also has applied-science potential as a biosensor or a biobattery. To this end, the enzyme has been efficiently immobilized on specific polymers and nanoparticles. This review spans the 6PGDH literature and all of the 6PGDH crystal structure data files held by the Protein Data Bank.

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

The 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.

Triosephosphate isomerase: the perfect enzyme, but how does it work?

Insights are offered on the study by Kelpsas et al. [IUCrJ (2021). 8, 633-643], who have combined neutron and X-ray crystallography then QM (quantum mechanics) calculations on triosephosphate isomerase (TIM). The authors dissect three possible enzyme mechanisms of TIM that have arisen in the decades since the first X-ray crystal structure of this enzyme was published in 1975.

X-ray crystallographic studies of RoAb13 bound to PIYDIN, a part of the N-terminal domain of C-C chemokine receptor 5.

C-C chemokine receptor 5 (CCR5) is a major co-receptor molecule used by HIV-1 to enter cells. This led to the hypothesis that stimulating an antibody response would block HIV with minimal toxicity. Here, X-ray crystallographic studies of the anti-CCR5 antibody RoAb13 together with two peptides were undertaken: one peptide is a 31-residue peptide containing the PIYDIN sequence and the other is the PIDYIN peptide alone, where PIYDIN is part of the N-terminal region of CCR5 previously shown to be important for HIV entry. In the presence of the longer peptide (the complete N-terminal domain), difference electron density was observed at a site within a hypervariable CDR3 binding region. In the presence of the shorter core peptide PIYDIN, difference electron density is again observed at this CDR3 site, confirming consistent binding for both peptides. This may be useful in the design of a new biomimetic to stimulate an antibody response to CCR5 in order to block HIV infection.

Combining X-rays, neutrons and electrons, and NMR, for precision and accuracy in structure-function studies.

The distinctive features of the physics-based probes used in understanding the structure of matter focusing on biological sciences, but not exclusively, are described in the modern context. This is set in a wider scope of holistic biology and the scepticism about `reductionism', what is called the `molecular level', and how to respond constructively. These topics will be set alongside the principles of accuracy and precision, and their boundaries. The combination of probes and their application together is the usual way of realizing accuracy. The distinction between precision and accuracy can be blurred by the predictive force of a precise structure, thereby lending confidence in its potential accuracy. These descriptions will be applied to the comparison of cryo and room-temperature protein crystal structures as well as the solid state of a crystal and the same molecules studied by small-angle X-ray scattering in solution and by electron microscopy on a sample grid. Examples will include: time-resolved X-ray Laue crystallography of an enzyme Michaelis complex formed directly in a crystal equivalent to in vivo; a new iodoplatin for radiation therapy predicted from studies of platin crystal structures; and the field of colouration of carotenoids, as an effective assay of function, i.e. their colouration, when unbound and bound to a protein. The complementarity of probes, as well as their combinatory use, is then at the foundation of real (biologically relevant), probe-artefacts-free, structure-function studies. The foundations of our methodologies are being transformed by colossal improvements in technologies of X-ray and neutron sources and their beamline instruments, as well as improved electron microscopes and NMR spectrometers. The success of protein structure prediction from gene sequence recently reported by CASP14 also opens new doors to change and extend the foundations of the structural sciences.

Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques.

This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26 Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65 Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug.