X-ray Diffraction Data Collection Research Articles

FDIP—A Fast Diffraction Image Processing Library for X-ray Crystallography Experiments

Serial crystallography (SX) is a cutting-edge technique in structural biology, involving the systematic collection of X-ray diffraction data from numerous randomly oriented microcrystals. To extract comprehensive three-dimensional information about the studied system, SX utilises thousands of measured diffraction patterns. As such, SX takes advantages of the properties of modern X-ray sources, including Free Electron Lasers (FELs) and third and fourth generation synchrotrons, as well as contemporary high-repetition-rate detectors. Efficient analysis of the extensive datasets generated during SX experiments demands fast and effective algorithms. The FDIP library offers meticulously optimised functions tailored for preprocessing data obtained in SX experiments. This encompasses tasks such as background subtraction, identification and masking of parasitic streaks, elimination of unwanted powder diffraction (e.g., from ice or salt crystals), and pinpointing useful Bragg peaks in each diffraction pattern. The library is equipped with a user-friendly graphical interface for facile parameter adjustment tailored to specific datasets. Compatible with popular SX processing software like OnDA, Cheetah, CrystFEL, and Merge3D, the FDIP library enhances the capabilities of these tools for streamlined and precise serial crystallography analyses.

Method—Setups and Protocols for Operando Batteries Experiments at Variable Temperatures Applied to CaxTiS2 and Na3−xV2(PO4)3

In this paper, we present two thermalized setups for in situ operando X-ray diffraction data collection in temperature ranging from 0° to 100 °C: a modified Leriche cell and a coin cell chamber. Both setups proved proper thermalization and representative electrochemistry. While the coin cell chamber may only be used in transmission mode, the modified Leriche cell can also be used in reflection mode. Two case studies are presented as example of operation: calcium insertion in TiS2 and sodium (de-)insertion in Na3−xV2(PO4)3. For the former, heating at 60 °C enables faster kinetics to form a solvent co-intercalated phase upon reduction, a process which is reversible upon reoxidation. For the later, Na3−xV2(PO4)3 is known to form a low temperature phase at ca 12 °C. Thanks to the thermalized setups, operando X-ray diffraction patterns were collected while cycling on the low temperature phase.

HpMCA: a Python-based graphical program for energy-dispersive X-ray diffraction data collection and analysis

ABSTRACT Energy-dispersive X-ray diffraction (EDXD) at synchrotron beamlines is commonly used for the study of material properties under high pressure and/or high temperature. Experimenters typically rely on the availability of robust data collection and analysis at a beamline, but this has become increasingly difficult, especially with the introduction of multi-element detectors that generate complex, multi-dimensional data sets. These data sets have energy resolution, and they can also be resolved in relation to sample position, diffraction angle, or different external stimuli. We report a new Python-based graphical program, hpMCA, for EDXD data collection and analysis that streamlines the experimental process for the beamline users. The program features a user-friendly interface, capability for online viewing and analyzing data from multi-element energy-dispersive detectors, and includes features useful for working with samples under high pressure and/or high temperature, such as crystal phase identification, real-time unit cell lattice refinement, and pressure determination based on an equation of state.

Crystallization of Poly(lactic acid) under continuous shear conditions: On-line x-ray analysis in isothermal conditions

In this work, the effect of shear rate on the isothermal crystallization of a poly(lactic acid), PLA, was analyzed. The crystallinity evolution of PLA during continuous shear was assessed by collecting X-ray diffraction data at short time intervals by adopting a synchrotron source. Shear rates in the range from 0.1 s−1 to 1.5 s−1 were analyzed. It was found that increasing the shear rate induced a dramatic decrease of the half crystallization times, more than one order of magnitude in the analyzed range of shear rates. A model for the description of crystallinity evolution was applied to describe the experimental results. It was found that the nucleation rate needed to describe the data are much larger than what measured in a previous work in the same range of shear rates but adopting a short step shear protocol.

Simultaneous quick-scanning X-ray absorption spectroscopy and X-ray diffraction

The existing setup for time-resolved quick-scanning X-ray absorption spectroscopy (QEXAFS) at beamline P64 at the PETRA III storage ring is complemented by new infrastructure suited for the collection of X-ray diffraction data simultaneously to the QEXAFS experiment. In particular, the sinusoidal periodic movements of the quick-EXAFS mono-chromator are modified in such a way, that an energy plateau with approximately constant energy over a certain time is created in the pre-edge region of the absorption edge of interest. By employing a properly defined trigger signal in this time, the exposure of a two-dimensional X-ray detector placed behind the sample is actuated, thus allowing to collect an X-ray diffraction pattern over a certain angular range as defined by the actual X-ray energy and the position of the 2D-detector. Details of the new setup as well as exemplary results obtained during the solvothermal synthesis of ZnO nanoparticles and the bcc-fcc phase transition of iron at elevated temperatures are presented.

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

Solid-State End-On to Side-On Isomerization of (N═N)2- in {[(R2N)3Nd]2N2}2- (R = SiMe3) Connects In Situ LnIII(NR2)3/K and Isolated [LnII(NR2)3]1- Dinitrogen Reduction.

Examination of the reduction chemistry of Nd(NR2)3 (R = SiMe3) under N2 has provided connections between the in situ Ln(III)-based LnIII(NR2)3/K reductions of N2 that form side-on bound neutral (N=N)2- complexes, [(R2N)2(THF)Ln]2[μ-η2:η2-N2], and the Ln(II)-based [LnII(NR2)3]1- reductions by Sc, Gd, and Tb that form end-on bound (N=N)2- complexes, {[(R2N)3Ln]2[μ-η1:η1-N2]}2-, which are dianions. The reduction of Nd(NR2)3 by KC8 under dinitrogen in Et2O in the presence of 18-crown-6 (18-c-6) forms dark yellow solutions of [K2(18-c-6)3]{[(R2N)3Nd]2N2} at low temperatures that become green as they warm up to -35 °C in a glovebox freezer. Green crystals obtained from the solution turn yellow-brown when cooled below -100 °C, and the yellow-brown compound has an end-on Nd2(μ-η1:η1-N2) structure. The yellow-brown crystals isomerize in the solid state on the diffractometer upon warming, and at -25 °C, the crystals are green and have a side-on Nd2(μ-η2:η2-N2) structure. Collection of X-ray diffraction data at 10 °C intervals from -50 to -90 °C revealed that the isomerization occurs at temperatures below -100 °C. In the presence of tetrahydrofuran (THF), the dianionic {[(R2N)3Nd]2N2}2- system can lose an amide ligand to provide the monoanionic [(R2N)3NdIII(μ-η2:η2-N2)NdIII(NR2)2(THF)]1-, characterized by X-ray crystallography. These data suggest a connection between the in situ Ln(III)/K reductions and Ln(II) reductions that depends on solvent, temperature, the presence of a chelate, and the specific rare-earth metal.

Expression, purification, crystallization and structure of Glyceraldehyde-3-Phosphate Dehydrogenase from Candida albicans, main causative agent of candidiasis

Glyceraldehyde-3-phosphate dehydrogenase from Candida albicans (CaGAPDH) is considered a therapeutic target for candidiasis. In this context, we report here the heterologous production, the three dimensional (3D) structure solution and some structural analyses and comparisons of CaGAPDH. The enzyme was expressed, purified and crystallized to collect X-ray diffraction data at the limit of 2.68 Å resolution, presenting some degree of translational non-crystallographic symmetry. Compared with other GAPDH sequences, CaGAPDH presents two paired mutations, Glu296 in place of a common Ala, and Ala258 in place of common Lys/Asn, which stand face to face, therefore, hydrophilic/hydrophobic residues in interchanged positions in CaGAPDH.

XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase

Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances. While recent publications have proposed a radical-based catalytic mechanism as well as novel sulfonate-based binding modes of MCR for its native substrates, the structure of the active state of MCR, as well as a complete characterization of the reaction, remain elusive. Previous attempts to structurally characterize the active MCR-Ni(I) state have been unsuccessful due to oxidation of the redox- sensitive catalytic Ni center. Further, while many cryo structures of the inactive Ni(II)-enzyme in various substrates-bound forms have been published, no room temperature structures have been reported, and the structure and mechanism of MCR under physiologically relevant conditions is not known. In this study, we report the first room temperature structure of the MCRred1-silent Ni(II) form using an X-ray Free-Electron Laser (XFEL), with simultaneous X-ray Emission Spectroscopy (XES) and X-ray Diffraction (XRD) data collection. In celebration of the seminal contributions of inorganic chemist Dick Holm to our understanding of nickel-based catalysis, we are honored to announce our findings in this special issue dedicated to this remarkable pioneer of bioinorganic chemistry.

2,5-Diketopiperazines via Intramolecular N-Alkylation of Ugi Adducts: A Contribution to the Synthesis, Density Functional Theory Study, X-ray Characterization, and Potential Herbicide Application

To investigate the herbicidal potential of 2,5-diketopiperazines (2,5-DKPs), we applied a known protocol to produce a series of 2,5-DKPs through intramolecular N-alkylation of Ugi adducts. However, the method was not successful for the cyclization of adducts presenting aromatic rings with some substituents at the ortho position. Results from DFT calculations showed that the presence of voluminous groups at the ortho position of a benzene ring results in destabilization of the transition structure. Lower activation enthalpies for the SN2-type cyclization of Ugi adducts were obtained when bromine, instead of a chlorine anion, is the leaving group, indicating that the activation enthalpy for the cyclization step controls the formation of the 2,5-DKP. Some Ugi adducts and 2,5-DKPs formed crystals with suitable qualities for single-crystal X-ray diffraction data collection. Phytotoxic damage of some 2,5-DKPs on leaves of the weed Euphorbia heterophylla did not differ from those caused by the commercial herbicide diquat.

Molecular Cloning and Purification of the Protein Lysine Methyltransferase SMYD2 and its Co-crystallization with a Target Peptide from Estrogen Receptor Alpha.

Methylation of estrogen receptor α by the protein lysine methyltransferase SMYD2 regulates ERα chromatin recruitment and its target gene expression. This protocol describes SMYD2 molecular cloning and purification and crystallization of SMYD2 in complex with an ERα peptide. Recombinant SMYD2 is constructed and overexpressed in Escherichia coli cells. After release from the cells by French Press, SMYD2 is purified to apparent homogeneity with multiple chromatography methods. Nickel affinity column purifies SMYD2 based on specific interaction of its 6xHis tag with the bead-immobilized nickel ions. Desalting column is used for protein buffer exchange. Gel filtration column purifies SMYD2 based on molecular size. The entire purification process is monitored and analyzed by SDS-polyacrylamide gel electrophoresis. Crystallization of SMYD2 is performed with the hanging-drop vapor diffusion method. Crystals of the SMYD2-ERα peptide complex are obtained by microseeding using Seeding Bead. This method can give rise to large size of crystals which are suitable for X-ray diffraction data collection. X-ray crystallographic study of the SMYD2-ERα complex can provide structural insight into posttranslational regulation of ERα signaling.

Multi-phase equation of state of ultrapure hafnium to 120 GPa

Hafnium (Hf) is an industrially important material due to its large neutron absorption cross-section and its high corrosion resistance. When subjected to high pressure, Hf phase transforms from its hexagonal close packed α-Hf phase to the hexagonal ω-Hf phase. Upon further compression, ω-Hf phase transforms to the body centered cubic β-Hf phase. In this study, the high pressure phase transformations of Hf are studied by compressing and decompressing a well-characterized Hf sample in diamond anvil cells up to 120 GPa while collecting x-ray diffraction data. The phase transformations of Hf were compared in both a He pressure transmitting medium (PTM) and no PTM over several experiments. It was found that the α-Hf to ω-Hf phase transition occurs at a higher pressure during compression and lower pressure during decompression with a helium (He) PTM compared to using no PTM. There was little difference in the ω-Hf to β-Hf phase transition pressure between the He PTM and no PTM. The equation of state was fit for all three phases of Hf and under both PTM and no-PTM.

Synthesis of drynaran and analogues

In a previous work, the natural compound drynaran, namely E-3-(5-hydroxymethyl)furan-2-yl)-2-phenylacrylaldehyde, was isolated from the Asian medicinal plant Drynaria bonii. Although this plant is used for several therapeutic purposes, drynaran was isolated in small quantities and, therefore, evaluated only for a few biological activities. Here, we propose a simple, fast, diastereoselective, and green synthesis of drynaran, using ethanol as solvent and 5-hydroxymethylfurfural as starting material. Drynaran was obtained in 66% yield and 98:2 E:Z diastereoisomeric ratio. The method was also used to produce a series of drynaran analogues, some of them as pure E-isomer and others in E:Z ratio of at least 90:10. Besides, five drynaran analogues formed crystals with suitable qualities for single-crystal X-ray diffraction data collection, and their crystal structures are described for the first time.

Equatorial aberration for powder diffraction data collected by continuous-scan integration of a silicon strip X-ray detector

The application of continuous-scan integration to collect X-ray diffraction data with a Si strip X-ray detector (CSI-SSXD) introduces additional effects on the peak shift and deformation of peak shape caused by the equatorial aberration. A deconvolutional method to correct the effects of equatorial aberration in CSI-SSXD data is proposed in this study. There are four critical angles related to the effects of spillover of the incident X-ray beam from the specimen face in the CSI-SSXD data. Exact values of cumulants of the equatorial aberration function are efficiently evaluated by 4 × 4 point two-dimensional Gauss–Legendre integral. A naïve two-step deconvolutional method has been applied to remove the effects of the first and third-order cumulants of the equatorial aberration function from the observed CSI-SSXD data. The performance of the algorithm has been tested by analyses of CSI-SSXD data of three LaB6 powder specimens with the widths of 20, 10, and 5 mm, collected with a diffractometer with the goniometer radius of 150 mm.

Chemical preparation, crystal structure reinvestigation, and vibrational study of meta-carboxyphenyl ammonium dihydrogenomonophosphate (C7H4NH3OOH)H2PO4 (m-C AMP)

Chemical preparation, X-ray diffraction data collection on a single crystal and IR spectroscopy studies of the monophosphate (C7H4NH3OOH)H2PO4, meta-carboxyphenyl ammonium dihydrogenophosphate crystal (m-CPAMP) were developped at room temperature. The title compound crystallizes in the monoclinic system, with the centrosymmetric space group P21/c (Z = 4). Its unit-cell dimensions are a = 12.9361 (7) (Å), b = 11.7735 (6) (Å), c = 6.5764 (4) (Å), β = 102.668 (2)° and V = 977.22 (9) Å3. The structure determined gives a clear description of the hydrogen bonds connecting the hydrogen phosphate H2PO4- with the organic matrix. In the structure of this compound, each (H2PO4)− aggregates with cation through hydrogen-bond interactions of O-H----O(P) and N-H····O(P) types, and with another (H2PO4)− via hydrogen bonding O1-H····O3, forming symmetrical (H4P2O8)2 − anionic units.The Infrared spectrum of (C7H4NH3OOH)H2PO4 compound is studied in comparison to that of the cation C7H4NH3OOH considered in its free symmetry.

Is the R index accurate to assess the preferred orientation of portlandite in cement pastes?

The evaluation of portlandite orientation through the R index obtained from X-ray diffraction (XRD) pattern has become popular in recent years, and many works related this index with the mechanical properties of the composite. Such index is defined by 1.35 times the ratio between the intensities of the 001 and 101 reflections XRD pattern. However, most of these works neither present specific strategies to minimize preferred orientation due to sample preparation nor account for experimental contribution in the background. This paper pointed some factors that affect XRD data collection and interpretation, and discussed their effects on R index determination. Powder XRD was conducted using different sample holders and collection times, in addition to in-situ measurements for fresh paste. The results showed that sample preparation itself results in important intrinsic variability, which can be mitigated in Rietveld refinement but not in R index analysis. In addition, the sample type played an important role in portlandite assessment: paste slice kept the random distribution of portlandite crystals but damaged ettringite, while powder press induces preferred orientation perpendicularly to the pressing direction. Powder backloading only partially reduced the preferred orientation issue. In-situ sample can keep the original matrix arrangement, but such analysis is difficult for long periods. Furthermore, a corrected R index is proposed, in order to consider the contribution of the background. Overall, the use of the R index for portlandite orientation assessment must be carefully addressed to avoid misinterpretations.

Structure of Enterohemorrhagic Escherichia coli O157:H7 Intimin Virulence Factor Bound to Nanobodies

Enterohemorrhagic E. coli O157:H7 (EHEC) is a food and water-born pathogen that presents a significant risk to human health worldwide. EHEC naturally resides in the intestinal tract of cattle as normal flora but can be transmitted to humans when contaminated food is ingested. Once EHEC invades and colonizes the human intestinal tract, it secretes Shiga-like toxins causing serious and potentially fatal gastrointestinal diseases. Unlike most foodborne bacterial diseases, antibiotics are not recommended for treatment of EHEC as antibiotics can induce enterotoxin release, increasing the risk of hemolytic uremic syndrome. There is currently no effective treatment or vaccine against enterohemorrhagic E. coli. EHEC pathogenicity involves intimin, a virulence factor which mediates bacterial colonization of the host GI tract. Due to its critical role in EHEC pathogenesis, intimin has attracted attention as an antibacterial drug target. Inhibiting the interaction of intimin with its cognate bacterial secreted receptor Tir could neutralize bacterial colonization of the gastrointestinal tract. One novel solution for treating EHEC infection comes from a unique class of antibodies known as nanobodies. Nanobodies are the antigen binding domain of heavy chain antibodies produced by the Camelid family. Intimin specific nanobodies were generated by immunizing a llama followed by phage display and selection for high affinity intimin binding nanobodies. The resultant intimin-specific nanobodies neutralized EHEC in vitro. We hypothesize that nanobodies specifically bind the Tir-binding domain of intimin in order to neutralizing EHEC infection. The goal of this research project is to determine the complex crystal structure of the nanobodies bound to intimin. The structure of nanobodies-intimin would show their protein-protein interaction binding site revealing key amino acids involved in binding. These residues could also be involved in the binding of intimin to its natural ligand, Tir. Thus, revealing such residues could form the bases for identifying and developing novel therapeutics and biosensors for treating and preventing EHEC pathogenesis.We show through size exclusion chromatography that nanobodies bind intimin and form stable complexes. We have set up crystal trails of five nanobodies (Int1, Int2, Int3, Int4, and IntN1) complexed to intimin. Two of the five complexed proteins, Int2-intimin and Int3-intimn formed crystals. The crystal condition for the two protein complexes Int2 and Int3 bound to intimin were optimized by varying the concentration of precipitants and pH range. Crystals of the protein complexes were sent to the Canadian Light Source for X-ray diffraction data collection. Diffraction measurements of Int2-intimin and Int3-intimin complexes were collected and processed in space groups P1 and P222, respectively. Currently, we are analyzing the structure by molecular replacement.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for &lt;i&gt;In Situ&lt;/i&gt; X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions.

Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper-oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.