Celebrating JSR's 30th anniversary: reminiscences of a Main Editor.

Reminiscences of one of the founding Main Editors of JSR on its 30th anniversary.

Crystallographic raw data, education and refereeing.

This article provides an overview of the preservation of raw diffraction data, then addresses the impact on future plans in the education and training of our community with respect to raw diffraction data and its potential reuse, and, thirdly presents the issue of referee access to the underpinning diffraction data and coordinates, as well as the Protein Data Bank Validation Report, in the review process of structural biology articles submitted for publication. Overall I pay tribute to the scientific achievements of Alex Wlodawer, who is also an ardent advocate of the importance of experimental data.

On the origin and variation of colors in lobster carapace.

The chemical basis of the blue-black to pink-orange color change on cooking of lobster, due to thermal denaturation of an astaxanthin-protein complex, α-crustacyanin, in the lobster carapace, has so far been elusive. Here, we investigate the relaxation of the astaxanthin pigment from its bound enolate form to its neutral hydroxyketone form, as origin of the spectral shift, by analyzing the response of UV-vis spectra of a water-soluble 3-hydroxy-4-oxo-ß-ionone model of astaxanthin to increases in pH, and by performing extensive quantum chemical calculations over a wide range of chemical conditions. The enolization of astaxanthin is consistent with the X-ray diffraction data of ß-crustacyanin (PDB code: ) whose crystals possess the distinct blue color. We find that enolate formation is possible within the protein environment and associated with a large bathochromic shift, thus offering a cogent explanation for the blue-black color and the response to thermal denaturation and revealing the chemistry of astaxanthin upon complex formation.

Experiences with making diffraction image data available: what metadata do we need to archive?

Recently, the IUCr (International Union of Crystallography) initiated the formation of a Diffraction Data Deposition Working Group with the aim of developing standards for the representation of raw diffraction data associated with the publication of structural papers. Archiving of raw data serves several goals: to improve the record of science, to verify the reproducibility and to allow detailed checks of scientific data, safeguarding against fraud and to allow reanalysis with future improved techniques. A means of studying this issue is to submit exemplar publications with associated raw data and metadata. In a recent study of the binding of cisplatin and carboplatin to histidine in lysozyme crystals under several conditions, the possible effects of the equipment and X-ray diffraction data-processing software on the occupancies and B factors of the bound Pt compounds were compared. Initially, 35.3 GB of data were transferred from Manchester to Utrecht to be processed with EVAL. A detailed description and discussion of the availability of metadata was published in a paper that was linked to a local raw data archive at Utrecht University and also mirrored at the TARDIS raw diffraction data archive in Australia. By making these raw diffraction data sets available with the article, it is possible for the diffraction community to make their own evaluation. This led to one of the authors of XDS (K. Diederichs) to re-integrate the data from crystals that supposedly solely contained bound carboplatin, resulting in the analysis of partially occupied chlorine anomalous electron densities near the Pt-binding sites and the use of several criteria to more carefully assess the diffraction resolution limit. General arguments for archiving raw data, the possibilities of doing so and the requirement of resources are discussed. The problems associated with a partially unknown experimental setup, which preferably should be available as metadata, is discussed. Current thoughts on data compression are summarized, which could be a solution especially for pixel-device data sets with fine slicing that may otherwise present an unmanageable amount of data.

Glucose-6-phosphate dehydrogenase and its 3D structures from crystallography and electron cryo-microscopy.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway. It has been extensively studied by biochemical and structural techniques. 13 X-ray crystal structures and five electron cryo-microscopy structures in the PDB are focused on in this topical review. Two F420-dependent glucose-6-phosphate dehydrogenase (FGD) structures are also reported. The significant differences between human and parasite G6PDs can be exploited to find selective drugs against infections such as malaria and leishmaniasis. Furthermore, G6PD is a prognostic marker in several cancer types and is also considered to be a tumour target. On the other hand, FGD is considered to be a target against Mycobacterium tuberculosis and possesses a high biotechnological potential in biocatalysis and bioremediation.

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.

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.

The evolution of raw data archiving and the growth of its importance in crystallography.

The hardware for data archiving has expanded capacities for digital storage enormously in the past decade or more. The IUCr evaluated the costs and benefits of this within an official working group which advised that raw data archiving would allow ground truth reproducibility in published studies. Consultations of the IUCr's Commissions ensued via a newly constituted standing advisory committee, the Committee on Data. At all stages, the IUCr financed workshops to facilitate community discussions and possible methods of raw data archiving implementation. The recent launch of the IUCrData journal's Raw Data Letters is a milestone in the implementation of raw data archiving beyond the currently published studies: it includes diffraction patterns that have not been fully interpreted, if at all. The IUCr 75th Congress in Melbourne included a workshop on raw data reuse, discussing the successes and ongoing challenges of raw data reuse. This article charts the efforts of the IUCr to facilitate discussions and plans relating to raw data archiving and reuse within the various communities of crystallography, diffraction and scattering.

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 crystal structure analysis of the relative binding of cisplatin and carboplatin in a mixture with histidine in a protein studied at 100 and 300 K with repeated X-ray irradiation.

The anticancer agents cisplatin and carboplatin bind to histidine in a protein. This crystal structure study at data-collection temperatures of 100 and 300 K examines their relative binding affinities to a histidine side chain and the effect of a high X-ray radiation dose of up to ∼1.8 MGy on the stability of the subsequent protein-Pt adducts. Cisplatin binding is visible at the histidine residue, but carboplatin binding is not. Five refined X-ray crystal structures are presented: one at 100 K as a reference and four at 300 K. The diffraction resolutions are 1.8, 2.0, 2.8, 2.9 and 3.5 Å.

Diffraction structural biology - a new horizon.

An introductory overview to the special issue papers on diffraction structural biology in this issue of the journal.

Experiences with archived raw diffraction images data: capturing cisplatin after chemical conversion of carboplatin in high salt conditions for a protein crystal.

The archiving of raw diffraction images data is the focus of an IUCr Diffraction Data Deposition Working Group (see http://forums.iucr.org/). Experience in archiving and sharing of raw diffraction images data in collaboration between Manchester and Utrecht Universities, studying the binding of the important anti-cancer agents, cisplatin and carboplatin to histidine in a protein, has recently been published. Subsequently, these studies have been expanded due to further analyses of each data set of raw diffraction images using the diffraction data processing program XDS. The raw diffraction images, measured at Manchester University, are available for download at Utrecht University and now also mirrored at the Tardis Raw Diffraction Data Archive in Australia. Thus a direct comparison of processed diffraction and derived protein model data from XDS with the published results has been made. The issue of conversion of carboplatin to cisplatin under a high chloride salt concentration has been taken up and a detailed crystallographic assessment is provided. Overall, these new structural chemistry research results are presented followed by a short summary of developing raw data archiving policy and practicalities as well as documenting the challenge of making appropriate and detailed recording of the metadata for crystallography.

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.

Structural studies of the effect that dimethyl sulfoxide (DMSO) has on cisplatin and carboplatin binding to histidine in a protein.

The anticancer complexes cisplatin and carboplatin target the DNA major groove, forming intrastrand and interstrand cross-links between guanine bases through their N7 atoms, causing distortion of the DNA helix and apoptotic cell death. A major side effect of these drugs is toxicity, which is caused via binding to many proteins in the body. A range of crystallographic studies have been carried out involving the cocrystallization of hen egg-white lysozyme (HEWL) as a test protein with cisplatin and carboplatin in aqueous and dimethyl sulfoxide (DMSO) conditions. Different cryoprotectants, glycerol and Paratone, were used for each of the cisplatin and carboplatin cocrystallization cases, while silicone oil was used for studies involving N-acetylglucosamine (NAG). Both cisplatin and carboplatin do not bind to HEWL in aqueous media on the timescales of the conditions used here, but upon addition of DMSO two molecules of cisplatin or carboplatin bind either side of His15, which is the only His residue in lysozyme and is assumed to be an imidazolyl anion or a chemical resonance moiety, i.e. both imidazole N atoms are chemically reactive. To identify the platinum-peak positions in the 'with DMSO conditions', anomalous scattering maps were calculated as a cross-check with the F(o) - F(c) OMIT maps. Platinum-occupancy σ values were established using three different software programs in each case. The use of EVAL15 to process all of the diffraction data sets provided a consistent platform for a large ensemble of data sets for the various protein and platinum-compound model refinements with REFMAC5 and then SHELXTL. Overall, this extensive set of crystallization and cryoprotectant conditions allowed a systematic evaluation of cisplatin and carboplatin binding to lysozyme as a test protein via detailed X-ray crystal structure characterizations. DMSO is used as a super-solvent for drug delivery as it is deemed to cause no effect upon drug binding. However, these results show that addition of DMSO causes the platinum anticancer drugs to bind to HEWL. This effect should be considered in toxicity assessments of these drugs and perhaps more widely.

Room-temperature X-ray diffraction studies of cisplatin and carboplatin binding to His15 of HEWL after prolonged chemical exposure.

The anticancer complexes cisplatin and carboplatin are known to bind to both the Nδ and the Nℇ atoms of His15 of hen egg-white lysozyme (HEWL) in the presence of dimethyl sulfoxide (DMSO). However, neither binds in aqueous media after 4 d of crystallization and crystal growth, suggesting that DMSO facilitates cisplatin/carboplatin binding to the N atoms of His15 by an unknown mechanism. Crystals of HEWL cocrystallized with cisplatin in both aqueous and DMSO media, of HEWL cocrystallized with carboplatin in DMSO medium and of HEWL cocrystallized with cisplatin and N-acetylglucosamine (NAG) in DMSO medium were stored for between seven and 15 months. X-ray diffraction studies of these crystals were carried out on a Bruker APEX II home-source diffractometer at room temperature. Room-temperature X-ray diffraction data collection removed the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants might have had on binding to the protein. Both cisplatin and carboplatin still bind to both the Nδ and Nℇ atoms of His15 in DMSO media as expected, but more detail for the cyclobutanedicarboxylate (CBDC) moiety of carboplatin was observed at the Nℇ binding site. However, two molecules of cisplatin were now observed to be bound to His15 in aqueous conditions. The platinum peak positions were identified using anomalous difference electron-density maps as a cross-check with Fo-Fc OMIT electron-density maps. The occupancies of each binding site were calculated using SHELXTL. These results show that over time cisplatin binds to both N atoms of His15 of HEWL in aqueous media, whereas this binding is speeded up in the presence of DMSO. The implication of cisplatin binding to proteins after a prolonged period of time is an important consideration for the length of treatment in patients who are given cisplatin.

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.