Doses for X-ray and electron diffraction: New features in RADDOSE-3D including intensity decay models.

New features in the dose estimation program RADDOSE-3D are summarised. They include the facility to enter a diffraction intensity decay model which modifies the "Diffraction Weighted Dose" output from a "Fluence Weighted Dose" to a "Diffraction-Decay Weighted Dose", a description of RADDOSE-ED for use in electron diffraction experiments, where dose is historically quoted in electrons/Å2 rather than in gray (Gy), and finally the development of a RADDOSE-3D GUI, enabling easy access to all the options available in the program.

Identifying and avoiding radiation damage in macromolecular crystallography.

Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.

Welcoming five new Co-editors.

Five new Co-editors are appointed to the Editorial Board of Acta Cryst. D - Structural Biology.

Radiation damage to biological macromolecules∗.

In this review, we describe recent research developments into radiation damage effects in macromolecular X-ray crystallography observed at synchrotrons and X-ray free electron lasers. Radiation damage in small molecule X-ray crystallography, small angle X-ray scattering experiments, microelectron diffraction, and single particle cryo-electron microscopy is briefly covered.

Raimond B. G. Ravelli (25 March 1968-30 June 2023).

Raimond B. G. Ravelli is remembered.

AlphaFold and the future of structural biology.

This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

AlphaFold and the future of structural biology.

This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.

AlphaFold and the future of structural biology.

This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.

Variability in X-ray induced effects in [Rh(COD)Cl]2 with changing experimental parameters.

X-ray characterisation methods have undoubtedly enabled cutting-edge advances in all aspects of materials research. Despite the enormous breadth of information that can be extracted from these techniques, the challenge of radiation-induced sample change and damage remains prevalent. This is largely due to the emergence of modern, high-intensity X-ray source technologies and the growing potential to carry out more complex, longer duration in situ or in operando studies. The tunability of synchrotron beamlines enables the routine application of photon energy-dependent experiments. This work explores the structural stability of [Rh(COD)Cl]2, a widely used catalyst and precursor in the chemical industry, across a range of beamline parameters that target X-ray energies of 8 keV, 15 keV, 18 keV and 25 keV, on a powder X-ray diffraction synchrotron beamline at room temperature. Structural changes are discussed with respect to absorbed X-ray dose at each experimental setting associated with the respective photon energy. In addition, the X-ray radiation hardness of the catalyst is discussed, by utilising the diffraction data collected at the different energies to determine a dose limit, which is often considered in protein crystallography and typically overlooked in small molecule crystallography. This work not only gives fundamental insight into how damage manifests in this organometallic catalyst, but will encourage careful consideration of experimental X-ray parameters before conducting diffraction on similar radiation-sensitive organometallic materials.

Resolving the subtle details of human DNA alkyltransferase lesion search and repair mechanism by single-molecule studies.

SignificanceWe directly visualize DNA translocation and lesion recognition by the O6-alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn2+ occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Our data support preferential cluster formation by AGT at alkyl lesions, suggesting a role of these clusters in stabilizing lesion-bound complexes. From our data, we derive a new model for the lesion search and repair mechanism of AGT.

Quantifying and comparing radiation damage in the Protein Data Bank.

Radiation damage remains one of the major bottlenecks to accurate structure solution in protein crystallography. It can induce structural and chemical changes in protein crystals, and is hence an important consideration when assessing the quality and biological veracity of crystal structures in repositories like the Protein Data Bank (PDB). However, detection of radiation damage artefacts has traditionally proved very challenging. To address this, here we introduce the Bnet metric. Bnet summarises in a single value the extent of damage suffered by a crystal structure by comparing the B-factor values of damage-prone and non-damage-prone atoms in a similar local environment. After validating that Bnet successfully detects damage in 23 different crystal structures previously characterised as damaged, we calculate Bnet values for 93,978 PDB crystal structures. Our metric highlights a range of damage features, many of which would remain unidentified by the other summary statistics typically calculated for PDB structures.

Heterotypic interactions drive antibody synergy against a malaria vaccine candidate.

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.

Submission of structural biology data for review purposes.

The editors discuss the submission of structural biology data.

High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells.

Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.

Radiation damage to biological samples: still a pertinent issue.

An understanding of radiation damage effects suffered by biological samples during structural analysis using both X-rays and electrons is pivotal to obtain reliable molecular models of imaged molecules. This special issue on radiation damage contains six papers reporting analyses of damage from a range of biophysical imaging techniques. For X-ray diffraction, an in-depth study of multi-crystal small-wedge data collection single-wavelength anomalous dispersion phasing protocols is presented, concluding that an absorbed dose of 5 MGy per crystal was optimal to allow reliable phasing. For small-angle X-ray scattering, experiments are reported that evaluate the efficacy of three radical scavengers using a protein designed to give a clear signature of damage in the form of a large conformational change upon the breakage of a disulfide bond. The use of X-rays to induce OH radicals from the radiolysis of water for X-ray footprinting are covered in two papers. In the first, new developments and the data collection pipeline at the NSLS-II high-throughput dedicated synchrotron beamline are described, and, in the second, the X-ray induced changes in three different proteins under aerobic and low-oxygen conditions are investigated and correlated with the absorbed dose. Studies in XFEL science are represented by a report on simulations of ultrafast dynamics in protic ionic liquids, and, lastly, a broad coverage of possible methods for dose efficiency improvement in modalities using electrons is presented. These papers, as well as a brief synopsis of some other relevant literature published since the last Journal of Synchrotron Radiation Special Issue on Radiation Damage in 2019, are summarized below.

Structural and Electronic Effects of X-ray Irradiation on Prototypical [M(COD)Cl]2 Catalysts.

X-ray characterization techniques are invaluable for probing material characteristics and properties, and have been instrumental in discoveries across materials research. However, there is a current lack of understanding of how X-ray-induced effects manifest in small molecular crystals. This is of particular concern as new X-ray sources with ever-increasing brilliance are developed. In this paper, systematic studies of X-ray-matter interactions are reported on two industrially important catalysts, [Ir(COD)Cl]2 and [Rh(COD)Cl]2, exposed to radiation in X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) experiments. From these complementary techniques, changes to structure, chemical environments, and electronic structure are observed as a function of X-ray exposure, allowing comparisons of stability to be made between the two catalysts. Radiation dose is estimated using recent developments to the RADDOSE-3D software for small molecules and applied to powder XRD and XPS experiments. Further insights into the electronic structure of the catalysts and changes occurring as a result of the irradiation are drawn from density functional theory (DFT). The techniques combined here offer much needed insight into the X-ray-induced effects in transition-metal catalysts and, consequently, their intrinsic stabilities. There is enormous potential to extend the application of these methods to other small molecular systems of scientific or industrial relevance.

Zinc determines dynamical properties and aggregation kinetics of human insulin.

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment, and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here, we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, in which the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced x-ray emission and x-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.