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.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures.

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.

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.

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.

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.

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.

High-Throughput PIXE as an Essential Quantitative Assay for Accurate Metalloprotein Structural Analysis: Development and Application.

Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.

X-ray radiation damage to biological samples: recent progress.

With the continuing development of beamlines for macromolecular crystallography (MX) over the last few years providing ever higher X-ray flux densities, it has become even more important to be aware of the effects of radiation damage on the resulting structures. Nine papers in this issue cover a range of aspects related to the physics and chemistry of the manifestations of this damage, as observed in both MX and small-angle X-ray scattering (SAXS) on crystals, solutions and tissue samples. The reports include measurements of the heating caused by X-ray irradiation in ruby microcrystals, low-dose experiments examining damage rates as a function of incident X-ray energy up to 30 keV on a metallo-enzyme using a CdTe detector of high quantum efficiency as well as a theoretical analysis of the gains predicted in diffraction efficiency using these detectors, a SAXS examination of low-dose radiation exposure effects on the dissociation of a protein complex related to human health, theoretical calculations describing radiation chemistry pathways which aim to explain the specific structural damage widely observed in proteins, investigation of radiation-induced damage effects in a DNA crystal, a case study on a metallo-enzyme where structural movements thought to be mechanism related might actually be radiation-damage-induced changes, and finally a review describing what X-ray radiation-induced cysteine modifications can teach us about protein dynamics and catalysis. These papers, along with some other relevant literature published since the last Journal of Synchrotron Radiation Radiation Damage special issue in 2017, are briefly summarized below.

The potential benefits of using higher X-ray energies for macromolecular crystallography.

Using X-ray energies higher than those normally used (5-15 keV) for macromolecular X-ray crystallography (MX) at synchrotron sources can theoretically increase the achievable signal as a function of dose and reduce the rate of radiation damage. In practice, a major stumbling block to the use of higher X-ray energy has been the reduced quantum efficiency of silicon detectors as the X-ray energy increases, but hybrid photon-counting CdTe detectors are optimized for higher X-ray energies, and their performance has been steadily improving. Here the potential advantages of using higher incident beam energy together with a CdTe detector for MX are explored, with a particular focus on the advantages that higher beam energies may have for MX experiments with microbeams or microcrystals. Monte Carlo simulations are presented here which for the first time include the efficiency responses of some available X-ray detectors, as well as the possible escape of photoelectrons from the sample and their entry from surrounding material. The results reveal a `sweet spot' at an incident X-ray energy of 26 keV, and show a greater than factor of two improvement in diffraction efficiency at this energy when using microbeams and microcrystals of 5 µm or less.

Radiation-damage investigation of a DNA 16-mer.

In macromolecular crystallography, a great deal of effort has been invested in understanding radiation-damage progression. While the sensitivity of protein crystals has been well characterized, crystals of DNA and of DNA-protein complexes have not thus far been studied as thoroughly. Here, a systematic investigation of radiation damage to a crystal of a DNA 16-mer diffracting to 1.8 Šresolution and held at 100 K, up to an absorbed dose of 45 MGy, is reported. The RIDL (Radiation-Induced Density Loss) automated computational tool was used for electron-density analysis. Both the global and specific damage to the DNA crystal as a function of dose were monitored, following careful calibration of the X-ray flux and beam profile. The DNA crystal was found to be fairly radiation insensitive to both global and specific damage, with half of the initial diffraction intensity being lost at an absorbed average diffraction-weighted dose, D1/2, of 19 MGy, compared with 9 MGy for chicken egg-white lysozyme crystals under the same beam conditions but at the higher resolution of 1.4 Å. The coefficient of sensitivity of the DNA crystal was 0.014 Å2 MGy-1, which is similar to that observed for proteins. These results imply that the significantly greater radiation hardness of DNA and RNA compared with protein observed in a DNA-protein complex and an RNA-protein complex could be due to scavenging action by the protein, thereby protecting the DNA and RNA in these studies. In terms of specific damage, the regions of DNA that were found to be sensitive were those associated with some of the bound calcium ions sequestered from the crystallization buffer. In contrast, moieties farther from these sites showed only small changes even at higher doses.

Structural knowledge or X-ray damage? A case study on xylose isomerase illustrating both.

Xylose isomerase (XI) is an industrially important metalloprotein studied for decades. Its reaction mechanism has been postulated to involve movement of the catalytic metal cofactor to several different conformations. Here, a dose-dependent approach was used to investigate the radiation damage effects on XI and their potential influence on the reaction mechanism interpreted from the X-ray derived structures. Radiation damage is still one of the major challenges for X-ray diffraction experiments and causes both global and site-specific damage. In this study, consecutive high-resolution data sets from a single XI crystal from the same wedge were collected at 100 K and the progression of radiation damage was tracked over increasing dose (0.13-3.88 MGy). The catalytic metal and its surrounding amino acid environment experience a build-up of free radicals, and the results show radiation-damage-induced structural perturbations ranging from an absolute metal positional shift to specific residue motions in the active site. The apparent metal movement is an artefact of global damage and the resulting unit-cell expansion, but residue motion appears to be driven by the dose. Understanding and identifying radiation-induced damage is an important factor in accurately interpreting the biological conclusions being drawn.

Absorbed dose calculations for macromolecular crystals: improvements to RADDOSE. Erratum.

Corrections to an equation and a figure in the paper by Paithankar et al. (2009). [J. Synchrotron Rad. 16, 152-162] are made.

X-ray radiation damage to biological macromolecules: further insights.

Despite significant progress made over more than 15 years of research, structural biologists are still grappling with the issue of radiation damage suffered by macromolecular crystals which is induced by the resultant radiation chemistry occurring during X-ray diffraction experiments. Further insights into these effects and the possible mitigation strategies for use in both diffraction and SAXS experiments are given in eight papers in this volume. In particular, damage during experimental phasing is addressed, scavengers for SAXS experiments are investigated, microcrystals are imaged, data collection strategies are optimized, specific damage to tyrosine residues is reexamined, and room temperature conformational heterogeneity as a function of dose is explored. The brief summary below puts these papers into perspective relative to other ongoing radiation damage research on macromolecules.

OH cleavage from tyrosine: debunking a myth.

During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses on the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C-O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr -OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr -OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the Protein Data Bank has established that Tyr -OH electron density loss is not generally a dominant damage pathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposed to account for instances of observable Tyr -OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to disulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported.

Development of tools to automate quantitative analysis of radiation damage in SAXS experiments.

Biological small-angle X-ray scattering (SAXS) is an increasingly popular technique used to obtain nanoscale structural information on macromolecules in solution. However, radiation damage to the samples limits the amount of useful data that can be collected from a single sample. In contrast to the extensive analytical resources available for macromolecular crystallography (MX), there are relatively few tools to quantitate radiation damage for SAXS, some of which require a significant level of manual characterization, with the potential of leading to conflicting results from different studies. Here, computational tools have been developed to automate and standardize radiation damage analysis for SAXS data. RADDOSE-3D, a dose calculation software utility originally written for MX experiments, has been extended to account for the cylindrical geometry of the capillary tube, the liquid composition of the sample and the attenuation of the beam by the capillary material to allow doses to be calculated for many SAXS experiments. Furthermore, a library has been written to visualize and explore the pairwise similarity of frames. The calculated dose for the frame at which three subsequent frames are determined to be dissimilar is defined as the radiation damage onset threshold (RDOT). Analysis of RDOTs has been used to compare the efficacy of radioprotectant compounds to extend the useful lifetime of SAXS samples. Comparison of the RDOTs shows that, for radioprotectant compounds at 5 and 10 mM concentration, glycerol is the most effective compound. However, at 1 and 2 mM concentrations, dithiothreitol (DTT) appears to be most effective. Our newly developed visualization library contains methods that highlight the unusual radiation damage results given by SAXS data collected using higher concentrations of DTT: these observations should pave the way to the development of more sophisticated frame merging strategies.

The topology, structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C.

BACKGROUND: Mutations in Lipopolysaccharide-induced tumour necrosis factor-α factor (LITAF) cause the autosomal dominant inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1C (CMT1C). LITAF encodes a 17 kDa protein containing an N-terminal proline-rich region followed by an evolutionarily-conserved C-terminal 'LITAF domain', which contains all reported CMT1C-associated pathogenic mutations. RESULTS: Here, we report the first structural characterisation of LITAF using biochemical, cell biological, biophysical and NMR spectroscopic approaches. Our structural model demonstrates that LITAF is a monotopic zinc-binding membrane protein that embeds into intracellular membranes via a predicted hydrophobic, in-plane, helical anchor located within the LITAF domain. We show that specific residues within the LITAF domain interact with phosphoethanolamine (PE) head groups, and that the introduction of the V144M CMT1C-associated pathogenic mutation leads to protein aggregation in the presence of PE. CONCLUSIONS: In addition to the structural characterisation of LITAF, these data lead us to propose that an aberrant LITAF-PE interaction on the surface of intracellular membranes contributes to the molecular pathogenesis that underlies this currently incurable disease.

Predicting the X-ray lifetime of protein crystals.

Radiation damage is a major cause of failure in macromolecular crystallography experiments. Although it is always best to evenly illuminate the entire volume of a homogeneously diffracting crystal, limitations of the available equipment and imperfections in the sample often require a more sophisticated targeting strategy, involving microbeams smaller than the crystal, and translations of the crystal during data collection. This leads to a highly inhomogeneous distribution of absorbed X-rays (i.e., dose). Under these common experimental conditions, the relationship between dose and time is nonlinear, making it difficult to design an experimental strategy that optimizes the radiation damage lifetime of the crystal, or to assign appropriate dose values to an experiment. We present, and experimentally validate, a predictive metric diffraction-weighted dose for modeling the rate of decay of total diffracted intensity from protein crystals in macromolecular crystallography, and hence we can now assign appropriate "dose" values to modern experimental setups. Further, by taking the ratio of total elastic scattering to diffraction-weighted dose, we show that it is possible to directly compare potential data-collection strategies to optimize the diffraction for a given level of damage under specific experimental conditions. As an example of the applicability of this method, we demonstrate that by offsetting the rotation axis from the beam axis by 1.25 times the full-width half maximum of the beam, it is possible to significantly extend the dose lifetime of the crystal, leading to a higher number of diffracted photons, better statistics, and lower overall radiation damage.

Radiation damage to macromolecules: kill or cure?

Radiation damage induced by X-ray beams during macromolecular diffraction experiments remains an issue of concern in structural biology. While advances in our understanding of this phenomenon, driven in part by a series of workshops in this area, undoubtedly have been and are still being made, there are still questions to be answered. Eight papers in this volume give a flavour of ongoing investigations, addressing various issues. These range over: a proposed new metric derived from atomic B-factors for identifying potentially damaged amino acid residues, a study of the relative damage susceptibility of protein and DNA in a DNA/protein complex, a report of an indication of specific radiation damage to a protein determined from data collected using an X-ray free-electron laser (FEL), an account of the challenges in FEL raw diffraction data analysis, an exploration of the possibilities of using radiation damage induced phasing to solve structures using FELs, simulations of radiation damage as a function of FEL temporal pulse profiles, results on the influence of radiation damage during scanning X-ray diffraction measurements and, lastly, consideration of strategies for minimizing radiation damage during SAXS experiments. In this short introduction, these contributions are briefly placed in the context of other current work on radiation damage in the field.

Identifying and quantifying radiation damage at the atomic level.

Radiation damage impedes macromolecular diffraction experiments. Alongside the well known effects of global radiation damage, site-specific radiation damage affects data quality and the veracity of biological conclusions on protein mechanism and function. Site-specific radiation damage follows a relatively predetermined pattern, in that different structural motifs are affected at different dose regimes: in metal-free proteins, disulfide bonds tend to break first followed by the decarboxylation of aspartic and glutamic acids. Even within these damage motifs the decay does not progress uniformly at equal rates. Within the same protein, radiation-induced electron density decay of a particular chemical group is faster than for the same group elsewhere in the protein: an effect known as preferential specific damage. Here, BDamage, a new atomic metric, is defined and validated to recognize protein regions susceptible to specific damage and to quantify the damage at these sites. By applying BDamage to a large set of known protein structures in a statistical survey, correlations between the rates of damage and various physicochemical parameters were identified. Results indicate that specific radiation damage is independent of secondary protein structure. Different disulfide bond groups (spiral, hook, and staple) show dissimilar radiation damage susceptibility. There is a consistent positive correlation between specific damage and solvent accessibility.