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A comparative anatomy of protein crystals: lessons from the automatic processing of 56 000 samples

The fully automatic processing of crystals of macromolecules has presented a unique opportunity to gather information on the samples that is not usually recorded. This has proved invaluable in improving sample-location, characterization and data-collection algorithms. After operating for four years, MASSIF-1 has now processed over 56 000 samples, gathering information at each stage, from the volume of the crystal to the unit-cell dimensions, the space group, the quality of the data collected and the reasoning behind the decisions made in data collection. This provides an unprecedented opportunity to analyse these data together, providing a detailed landscape of macromolecular crystals, intimate details of their contents and, importantly, how the two are related. The data show that mosaic spread is unrelated to the size or shape of crystals and demonstrate experimentally that diffraction intensities scale in proportion to crystal volume and molecular weight. It is also shown that crystal volume scales inversely with molecular weight. The results set the scene for the development of X-ray crystallography in a changing environment for structural biology.




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Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV–visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15–40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.




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Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography

Copper-containing nitrite reductases (CuNiRs) that convert NO2− to NO via a CuCAT–His–Cys–CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD−, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2− a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.




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1 kHz fixed-target serial crystallography using a multilayer monochromator and an integrating pixel detector

Reliable sample delivery and efficient use of limited beam time have remained bottlenecks for serial crystallography (SX). Using a high-intensity polychromatic X-ray beam in combination with a newly developed charge-integrating JUNGFRAU detector, we have applied the method of fixed-target SX to collect data at a rate of 1 kHz at a synchrotron-radiation facility. According to our data analysis for the given experimental conditions, only about 3 000 diffraction patterns are required for a high-quality diffraction dataset. With indexing rates of up to 25%, recording of such a dataset takes less than 30 s.




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Charge densities in actinide compounds: strategies for data reduction and model building

The data quality requirements for charge density studies on actinide compounds are extreme. Important steps in data collection and reduction required to obtain such data are summarized and evaluated. The steps involved in building an augmented Hansen–Coppens multipole model for an actinide pseudo-atom are provided. The number and choice of radial functions, in particular the definition of the core, valence and pseudo-valence terms are discussed. The conclusions in this paper are based on a re-examination and improvement of a previously reported study on [PPh4][UF6]. Topological analysis of the total electron density shows remarkable agreement between experiment and theory; however, there are significant differences in the Laplacian distribution close to the uranium atoms which may be due to the effective core potential employed for the theoretical calculations.




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Conformational characterization of full-length X-chromosome-linked inhibitor of apoptosis protein (XIAP) through an integrated approach

The X-chromosome-linked inhibitor of apoptosis protein (XIAP) is a multidomain protein whose main function is to block apoptosis by caspase inhibition. XIAP is also involved in other signalling pathways, including NF-κB activation and copper homeostasis. XIAP is overexpressed in tumours, potentiating cell survival and resistance to chemotherapeutics, and has therefore become an important target for the treatment of malignancy. Despite the fact that the structure of each single domain is known, the conformation of the full-length protein has never been determined. Here, the first structural model of the full-length XIAP dimer, determined by an integrated approach using nuclear magnetic resonance, small-angle X-ray scattering and electron paramagnetic resonance data, is presented. It is shown that XIAP adopts a compact and relatively rigid conformation, implying that the spatial arrangement of its domains must be taken into account when studying the interactions with its physiological partners and in developing effective inhibitors.




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A cryo-EM grid preparation device for time-resolved structural studies

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.




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A comparative study of single-particle cryo-EM with liquid-nitrogen and liquid-helium cooling

Radiation damage is the most fundamental limitation for achieving high resolution in electron cryo-microscopy (cryo-EM) of biological samples. The effects of radiation damage are reduced by liquid-helium cooling, although the use of liquid helium is more challenging than that of liquid nitrogen. To date, the benefits of liquid-nitrogen and liquid-helium cooling for single-particle cryo-EM have not been compared quantitatively. With recent technical and computational advances in cryo-EM image recording and processing, such a comparison now seems timely. This study aims to evaluate the relative merits of liquid-helium cooling in present-day single-particle analysis, taking advantage of direct electron detectors. Two data sets for recombinant mouse heavy-chain apoferritin cooled with liquid-nitrogen or liquid-helium to 85 or 17 K were collected, processed and compared. No improvement in terms of resolution or Coulomb potential map quality was found for liquid-helium cooling. Interestingly, beam-induced motion was found to be significantly higher with liquid-helium cooling, especially within the most valuable first few frames of an exposure, thus counteracting any potential benefit of better cryoprotection that liquid-helium cooling may offer for single-particle cryo-EM.




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High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.




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CryoEM at 100 keV: a demonstration and prospects

100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200 keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300 Å in thickness, the predicted optimal electron energy for imaging is 100 keV. Currently available electron cryomicroscopes in the 100–120 keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100 keV electrons. To demonstrate the potential of imaging at 100 kV, the voltage of a standard, commercial 200 kV field-emission gun (FEG) microscope was reduced to 100 kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100 keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5 MDa to 64 kDa with corresponding diameters from 320 to 72 Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4 Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100 keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.




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Throughput and resolution with a next-generation direct electron detector

Direct electron detectors (DEDs) have revolutionized cryo-electron microscopy (cryo-EM) by facilitating the correction of beam-induced motion and radiation damage, and also by providing high-resolution image capture. A new-generation DED, the DE64, has been developed by Direct Electron that has good performance in both integrating and counting modes. The camera has been characterized in both modes in terms of image quality, throughput and resolution of cryo-EM reconstructions. The modulation transfer function, noise power spectrum and detective quantum efficiency (DQE) were determined for both modes, as well as the number of images per unit time. Although the DQE for counting mode was superior to that for integrating mode, the data-collection throughput for this mode was more than ten times slower. Since throughput and resolution are related in single-particle cryo-EM, data for apoferritin were collected and reconstructed using integrating mode, integrating mode in conjunction with a Volta phase plate (VPP) and counting mode. Only the counting-mode data resulted in a better than 3 Å resolution reconstruction with similar numbers of particles, and this increased performance could not be compensated for by the increased throughput of integrating mode or by the increased low-frequency contrast of integrating mode with the VPP. These data show that the superior image quality provided by counting mode is more important for high-resolution cryo-EM reconstructions than the superior throughput of integrating mode.




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A fixed-target platform for serial femtosecond crystallography in a hydrated environment

For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.




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The resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase

Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.




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Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1

Methods are presented that detect three types of aberrations in single-particle cryo-EM data sets: symmetrical and antisymmetrical optical aberrations and magnification anisotropy. Because these methods only depend on the availability of a preliminary 3D reconstruction from the data, they can be used to correct for these aberrations for any given cryo-EM data set, a posteriori. Using five publicly available data sets, it is shown that considering these aberrations improves the resolution of the 3D reconstruction when these effects are present. The methods are implemented in version 3.1 of the open-source software package RELION.




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On the mechanism of solid-state phase transitions in molecular crystals – the role of cooperative motion in (quasi)racemic linear amino acids

During single-crystal-to-single-crystal (SCSC) phase transitions, a polymorph of a compound can transform to a more stable form while remaining in the solid state. By understanding the mechanism of these transitions, strategies can be developed to control this phenomenon. This is particularly important in the pharmaceutical industry, but also relevant for other industries such as the food and agrochemical industries. Although extensive literature exists on SCSC phase transitions in inorganic crystals, it is unclear whether their classications and mechanisms translate to molecular crystals, with weaker interactions and more steric hindrance. A comparitive study of SCSC phase transitions in aliphatic linear-chain amino acid crystals, both racemates and quasi-racemates, is presented. A total of 34 transitions are considered and most are classified according to their structural change during the transition. Transitions without torsional changes show very different characteristics, such as transition temperature, enthalpy and free energy, compared with transitions that involve torsional changes. These differences can be rationalized using classical nucleation theory and in terms of a difference in mechanism; torsional changes occur in a molecule-by-molecule fashion, whereas transitions without torsional changes involve cooperative motion with multiple molecules at the same time.




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High-resolution cryo-EM reconstructions in the presence of substantial aberrations

Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.




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Fast and accurate defocus modulation for improved tunability of cryo-EM experiments

Current data collection strategies in electron cryo-microscopy (cryo-EM) record multiframe movies with static optical settings. This limits the number of adjustable parameters that can be used to optimize the experiment. Here, a method for fast and accurate defocus (FADE) modulation during movie acquisition is proposed. It uses the objective lens aperture as an electrostatic pole that locally modifies the electron beam potential. The beam potential variation is converted to defocus change by the typically undesired chromatic aberration of the objective lens. The simplicity, electrostatic principle and low electrical impedance of the device allow fast switching speeds that will enable per-frame defocus modulation of cryo-EM movies. Researchers will be able to define custom defocus `recipes' and tailor the experiment for optimal information extraction from the sample. The FADE method could help to convert the microscope into a more dynamic and flexible optical platform that delivers better performance in cryo-EM single-particle analysis and electron cryo-tomography.




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A decagonal quasicrystal with rhombic and hexagonal tiles decorated with icosahedral structural units

The structure of a decagonal quasicrystal in the Zn58Mg40Y2 (at.%) alloy was studied using electron diffraction and atomic resolution Z-contrast imaging techniques. This stable Frank–Kasper Zn–Mg–Y decagonal quasicrystal has an atomic structure which can be modeled with a rhombic/hexagonal tiling decorated with icosahedral units at each vertex. No perfect decagonal clusters were observed in the Zn–Mg–Y decagonal quasicrystal, which differs from the Zn–Mg–Dy decagonal crystal with the same space group P10/mmm. Y atoms occupy the center of `dented decagon' motifs consisting of three fat rhombic and two flattened hexagonal tiles. About 75% of fat rhombic tiles are arranged in groups of five forming star motifs, while the others connect with each other in a `zigzag' configuration. This decagonal quasicrystal has a composition of Zn68.3Mg29.1Y2.6 (at.%) with a valence electron concentration (e/a) of about 2.03, which is in accord with the Hume–Rothery criterion for the formation of the Zn-based quasicrystal phase (e/a = 2.0–2.15).




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Structures of substrate- and product-bound forms of a multi-domain copper nitrite reductase shed light on the role of domain tethering in protein complexes

Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitro­gen cycle where nitrate is used in place of di­oxy­gen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering than the intuitive advantage for a partner-protein electron-transfer complex by narrowing the conformational search in such a combined system.




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Biochemical and structural explorations of α-hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallo­graphy, were exploited to reach these conclusions and provide additional insights.




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Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal unit-cell packing reveals different crystallographic and noncrystallographic dimers of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.




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Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp–Glu–Ser), Nematoda (Asp–Asp–His) and Echinodermata (Asp–Glu–Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.




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Structures of the substrate-binding protein YfeA in apo and zinc-reconstituted holo forms

In the structural biology of bacterial substrate-binding proteins (SBPs), a growing number of comparisons between substrate-bound and substrate-free forms of metal atom-binding (cluster A-I) SBPs have revealed minimal structural differences between forms. These observations contrast with SBPs that bind substrates such as amino acids or nucleic acids and may undergo >60° rigid-body rotations. Substrate transfer in these SBPs is described by a Venus flytrap model, although this model may not apply to all SBPs. In this report, structures are presented of substrate-free (apo) and reconstituted substrate-bound (holo) YfeA, a polyspecific cluster A-I SBP from Yersinia pestis. It is demonstrated that an apo cluster A-I SBP can be purified by fractionation when co-expressed with its cognate transporter, adding an alternative strategy to the mutagenesis or biochemical treatment used to generate other apo cluster A-I SBPs. The apo YfeA structure contains 111 disordered protein atoms in a mobile helix located in the flexible carboxy-terminal lobe. Metal binding triggers a 15-fold reduction in the solvent-accessible surface area of the metal-binding site and reordering of the 111 protein atoms in the mobile helix. The flexible lobe undergoes a 13.6° rigid-body rotation that is driven by a spring-hammer metal-binding mechanism. This asymmetric rigid-body rotation may be unique to metal atom-binding SBPs (i.e. clusters A-I, A-II and D-IV).




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The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.




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Controlled dehydration, structural flexibility and gadolinium MRI contrast compound binding in the human plasma glycoprotein afamin

Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an ∼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.




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Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.




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Improved chemistry restraints for crystallographic refinement by integrating the Amber force field into Phenix

The refinement of biomolecular crystallographic models relies on geometric restraints to help to address the paucity of experimental data typical in these experiments. Limitations in these restraints can degrade the quality of the resulting atomic models. Here, an integration of the full all-atom Amber molecular-dynamics force field into Phenix crystallographic refinement is presented, which enables more complete modeling of biomolecular chemistry. The advantages of the force field include a carefully derived set of torsion-angle potentials, an extensive and flexible set of atom types, Lennard–Jones treatment of nonbonded interactions and a full treatment of crystalline electrostatics. The new combined method was tested against conventional geometry restraints for over 22 000 protein structures. Structures refined with the new method show substantially improved model quality. On average, Ramachandran and rotamer scores are somewhat better, clashscores and MolProbity scores are significantly improved, and the modeling of electrostatics leads to structures that exhibit more, and more correct, hydrogen bonds than those refined using traditional geometry restraints. In general it is found that model improvements are greatest at lower resolutions, prompting plans to add the Amber target function to real-space refinement for use in electron cryo-microscopy. This work opens the door to the future development of more advanced applications such as Amber-based ensemble refinement, quantum-mechanical representation of active sites and improved geometric restraints for simulated annealing.




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What is the structural chemistry of the living organism at its temperature and pressure?

The three probes of the structure of matter (X-rays, neutrons and electrons) in biology have complementary properties and strengths. The balance between these three probes within their strengths and weaknesses is perceived to change, even dramatically so at times. For the study of combined states of order and disorder, NMR crystallography is also applicable. Of course, to understand biological systems the required perspectives are surely physiologically relevant temperatures and relevant chemical conditions, as well as a minimal perturbation owing to the needs of the probe itself. These remain very tough challenges because, for example, cryoEM by its very nature will never be performed at room temperature, crystallization often requires nonphysiological chemical conditions, and X-rays and electrons cause beam damage. However, integrated structural biology techniques and functional assays provide a package towards physiological relevance of any given study. Reporting of protein crystal structures, and their associated database entries, could usefully indicate how close to the biological situation they are, as discussed in detail in this feature article.




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ALEPH: a network-oriented approach for the generation of fragment-based libraries and for structure interpretation

The analysis of large structural databases reveals general features and relationships among proteins, providing useful insight. A different approach is required to characterize ubiquitous secondary-structure elements, where flexibility is essential in order to capture small local differences. The ALEPH software is optimized for the analysis and the extraction of small protein folds by relying on their geometry rather than on their sequence. The annotation of the structural variability of a given fold provides valuable information for fragment-based molecular-replacement methods, in which testing alternative model hypotheses can succeed in solving difficult structures when no homology models are available or are successful. ARCIMBOLDO_BORGES combines the use of composite secondary-structure elements as a search model with density modification and tracing to reveal the rest of the structure when both steps are successful. This phasing method relies on general fold libraries describing variations around a given pattern of β-sheets and helices extracted using ALEPH. The program introduces characteristic vectors defined from the main-chain atoms as a way to describe the geometrical properties of the structure. ALEPH encodes structural properties in a graph network, the exploration of which allows secondary-structure annotation, decomposition of a structure into small compact folds, generation of libraries of models representing a variation of a given fold and finally superposition of these folds onto a target structure. These functions are available through a graphical interface designed to interactively show the results of structure manipulation, annotation, fold decomposition, clustering and library generation. ALEPH can produce pictures of the graphs, structures and folds for publication purposes.




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From space group to space groupoid: the partial symmetry of low-temperature E-vanillyl oxime

The phase transition of E-vanillyl oxime {1-[(E)-(hydroxyimino)methyl]-4-hydroxy-3-methoxybenzene, C8H9NO3} has been analysed by single-crystal and powder X-ray diffraction. The high-temperature (HT) phase (P21/a, Z' = 1) transforms into the low-temperature (LT) phase (threefold superstructure, Poverline{1}, Z' = 6) at ca 190 K. The point operations lost on cooling, {m[010], 2[010]}, are retained as twin operations and constitute the twin law. The screw rotations and glide reflections are retained in the LT phase as partial operations acting on a subset of Euclidean space {b E}^3. The full symmetry of the LT phase, including partial operations, is described by a disconnected space groupoid which is built of three connected components.




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Pristine and hydrated fluoroapatite (0001)

The surface structure of fluoro­apatite (0001) (FAp0001) under quasi-dry and humid conditions has been probed with surface X-ray diffraction (SXRD). Lateral and perpendicular atomic relaxations corresponding to the FAp0001 termination before and after H2O exposure and the location of the adsorbed water molecules have been determined from experimental analysis of the crystal truncation rod (CTR) intensities. The surface under dry conditions exhibits a bulk termination with relaxations in the outermost atomic layers. The hydrated surface is formed by a disordered partially occupied H2O layer containing one water molecule (33% surface coverage) adsorbed at each of the three surface Ca atoms, and is coupled with one OH group randomly bonded to each of the three topmost P atoms with a 33% surface coverage.




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On the puzzling case of sodium saccharinate 1.875-hydrate: structure description in (3+1)-dimensional superspace

The structure of sodium saccharinate 1.875-hydrate is presented in three- and (3+1)-dimensional space. The present model is more accurate than previously published superstructures, due to an excellent data set collected up to a high resolution of 0.89 Å−1. The present study confirms the unusual complexity of the structure comprising a very large primitive unit cell with Z' = 16. A much smaller degree of correlated disorder of parts of the unit cell is found than is present in the previously published models. As a result of pseudo-symmetry, the structure can be described in a higher-dimensional space. The X-ray diffraction data clearly indicate a (3+1)-dimensional periodic structure with stronger main reflections and weaker superstructure reflections. Furthermore, the structure is established as being commensurate. The structure description in superspace results in a four times smaller unit cell with an additional base centring of the lattice, resulting in an eightfold substructure (Z' = 2) of the 3D superstructure. Therefore, such a superspace approach is desirable to work out this high-Z' structure. The displacement and occupational modulation of the saccharinate anions have been studied, as well as their conformational variation along the fourth dimension.




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The influence of deuteration on the crystal structure of hybrid halide perovskites: a temperature-dependent neutron diffraction study of FAPbBr3

This paper discusses the full structural solution of the hybrid perovskite formamidinium lead tribromide (FAPbBr3) and its temperature-dependent phase transitions in the range from 3 K to 300 K using neutron powder diffraction and synchrotron X-ray diffraction. Special emphasis is put on the influence of deuteration on formamidinium, its position in the unit cell and disordering in comparison to fully hydrogenated FAPbBr3. The temperature-dependent measurements show that deuteration critically influences the crystal structures, i.e. results in partially-ordered temperature-dependent structural modifications in which two symmetry-independent molecule positions with additional dislocation of the molecular centre atom and molecular angle inclinations are present.




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Comparative study of the around-Fermi electronic structure of 5d metals and metal-oxides by means of high-resolution X-ray emission and absorption spectroscopies

The composition of occupied and unoccupied electronic states in the vicinity of Fermi energies is vital for all materials and relates to their physical, chemical and mechanical properties. This work demonstrates how the combination of resonant and non-resonant X-ray emission spectroscopies supplemented with theoretical modelling allows for quantitative analysis of electronic states in 5d transition metal and metal-oxide materials. Application of X-rays provides element selectivity that, in combination with the penetrating properties of hard X-rays, allows determination of the composition of electronic states under working conditions, i.e. non-vacuum environment. Tungsten metal and tungsten oxide are evaluated to show the capability to simultaneously assess composition of around-band-gap electronic states as well as the character and magnitude of the crystal field splitting.




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High-efficiency ultra-precision comparator for d-spacing mapping measurement of silicon

This article describes a high-efficiency experimental configuration for a self-referenced lattice comparator with a `brush beam' of synchrotron radiation from a bending magnet and two linear position-sensitive photon-counting-type X-ray detectors. The efficiency is more than ten times greater compared with the `pencil-beam' configuration and a pair of zero-dimensional detectors. A solution for correcting the systematic deviation of d-spacing measurements caused by the horizontal non-uniformity of the brush beam is provided. Also, the use of photon-counting-type one-dimensional detectors not only improves the spatial resolution of the measurements remarkably but can also adjust the sample's attitude angles easily.




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Saturation and self-absorption effects in the angle-dependent 2p3d resonant inelastic X-ray scattering spectra of Co3+

It is shown that the 2p3d resonant inelastic X-ray scattering intensity is distorted by saturation and self-absorption effects, i.e. by incident-energy-dependent saturation and by emission-energy-dependent self-absorption.




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A high-power, high-repetition-rate THz source for pump–probe experiments at Linac Coherent Light Source II




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3D grain reconstruction from laboratory diffraction contrast tomography

A method for reconstructing the three-dimensional grain structure from data collected with a recently introduced laboratory-based X-ray diffraction contrast tomography system is presented. Diffraction contrast patterns are recorded in Laue-focusing geometry. The diffraction geometry exposes shape information within recorded diffraction spots. In order to yield the three-dimensional crystallographic microstructure, diffraction spots are extracted and fed into a reconstruction scheme. The scheme successively traverses and refines solution space until a reasonable reconstruction is reached. This unique reconstruction approach produces results efficiently and fast for well suited samples.




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A temperature-controlled cold-gas humidifier and its application to protein crystals with the humid-air and glue-coating method

The room-temperature experiment has been revisited for macromolecular crystallography. Despite being limited by radiation damage, such experiments reveal structural differences depending on temperature, and it is expected that they will be able to probe structures that are physiologically alive. For such experiments, the humid-air and glue-coating (HAG) method for humidity-controlled experiments is proposed. The HAG method improves the stability of most crystals in capillary-free experiments and is applicable at both cryogenic and ambient temperatures. To expand the thermal versatility of the HAG method, a new humidifier and a protein-crystal-handling workbench have been developed. The devices provide temperatures down to 4°C and successfully maintain growth at that temperature of bovine cytochrome c oxidase crystals, which are highly sensitive to temperature variation. Hence, the humidifier and protein-crystal-handling workbench have proved useful for temperature-sensitive samples and will help reveal temperature-dependent variations in protein structures.




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Correlative vibrational spectroscopy and 2D X-ray diffraction to probe the mineralization of bone in phosphate-deficient mice

Bone crystallite chemistry and structure change during bone maturation. However, these properties of bone can also be affected by limited uptake of the chemical constituents of the mineral by the animal. This makes probing the effect of bone-mineralization-related diseases a complicated task. Here it is shown that the combination of vibrational spectroscopy with two-dimensional X-ray diffraction can provide unparalleled information on the changes in bone chemistry and structure associated with different bone pathologies (phosphate deficiency) and/or health conditions (pregnancy, lactation). Using a synergistic analytical approach, it was possible to trace the effect that changes in the remodelling regime have on the bone mineral chemistry and structure in normal and mineral-deficient (hypophosphatemic) mice. The results indicate that hypophosphatemic mice have increased bone remodelling, increased carbonate content and decreased crystallinity of the bone mineral, as well as increased misalignment of crystallites within the bone tissue. Pregnant and lactating mice that are normal and hypophosphatemic showed changes in the chemistry and misalignment of the apatite crystals that can be related to changes in remodelling rates associated with different calcium demand during pregnancy and lactation.




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POWGEN: rebuild of a third-generation powder diffractometer at the Spallation Neutron Source

The neutron powder diffractometer POWGEN at the Spallation Neutron Source has recently (2017–2018) undergone an upgrade which resulted in an increased detector complement along with a full overhaul of the structural design of the instrument. The current instrument has a solid angular coverage of 1.2 steradians and maintains the original third-generation concept, providing a single-histogram data set over a wide d-spacing range and high resolution to access large unit cells, detailed structural refinements and in situ/operando measurements.




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Successful sample preparation for serial crystallography experiments

Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.




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Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers. Corrigendum

Errors in the article by Opara, Martiel, Arnold, Braun, Stahlberg, Makita, David & Padeste [J. Appl. Cryst. (2017), 50, 909–918] are corrected.




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Detailed surface analysis of V-defects in GaN films on patterned silicon(111) substrates by metal–organic chemical vapour deposition. Corrigendum

An error in the article by Gao, Zhang, Zhu, Wu, Mo, Pan, Liu & Jiang [J. Appl. Cryst. (2019), 52, 637–642] is corrected.




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A temperature-controlled cold-gas humidifier and its application to protein crystals with the humid-air and glue-coating method

A new temperature-controllable humidifier for X-ray diffraction has been developed. It is shown that the humidifier can successfully maintain protein crystal growth at a temperature lower than room temperature.




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The competition between cocrystallization and separated crystallization based on crystallization from solution

Because researchers do not understand the formation mechanism of cocrystals, the preparation of cocrystals is mostly done by trial and error. This study focuses on the cocrystal formation mechanism to improve the efficiency of cocrystal preparation.




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A study of the strain distribution by scanning X-ray diffraction on GaP/Si for III–V monolithic integration on silicon

The distribution of plastic relaxation defects is studied using a nondestructive sub-micrometre X-ray diffraction scanning technique.




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Optimization of crystallization of biological macromolecules using dialysis combined with temperature control

This article describes rational strategies for the optimization of crystal growth using precise in situ control of the temperature and chemical composition of the crystallization solution through dialysis, to generate crystals of the specific sizes required for different downstream structure determination approaches.




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Accurate high-resolution single-crystal diffraction data from a Pilatus3 X CdTe detector

Detailed analysis of the high-flux deficiencies of pixel-array detectors leads to a protocol for the measurement of structure factors of unprecedented accuracy even for inorganic materials, and this significantly advances the prospects for experimental electron-density investigations.




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A thermal-gradient approach to variable-temperature measurements resolved in space

A new approach to variable-temperature measurements is presented, where the sample temperature changes continuously as a function of position.