Crystallography Experiments Research Articles

KINNTREX: a neural network to unveil protein mechanisms from time-resolved X-ray crystallography.

Here, a machine-learning method based on a kinetically informed neural network (NN) is introduced. The proposed method is designed to analyze a time series of difference electron-density maps from a time-resolved X-ray crystallographic experiment. The method is named KINNTREX (kinetics-informed NN for time-resolved X-ray crystallography). To validate KINNTREX, multiple realistic scenarios were simulated with increasing levels of complexity. For the simulations, time-resolved X-ray data were generated that mimic data collected from the photocycle of the photoactive yellow protein. KINNTREX only requires the number of intermediates and approximate relaxation times (both obtained from a singular valued decomposition) and does not require an assumption of a candidate mechanism. It successfully predicts a consistent chemical kinetic mechanism, together with difference electron-density maps of the intermediates that appear during the reaction. These features make KINNTREX attractive for tackling a wide range of biomolecular questions. In addition, the versatility of KINNTREX can inspire more NN-based applications to time-resolved data from biological macromolecules obtained by other methods.

Tethered Helical Ladder-Type Aromatic Lactams.

Tethered nonplanar aromatics (TNAs) make up an important class of nonplanar aromatic compounds showing unique features. However, the knowledge on the synthesis, structures, and properties of TNAs remains insufficient. In this work, a new type of TNAs, the tethered aromatic lactams, is synthesized via Pd-catalyzed consecutive intramolecular direct arylations. These molecules possess a helical ladder-type conjugated system of up to 13 fused rings. The overall yields ranged from 3.4 to 4.3%. The largest of the tethered aromatic lactams, 6L-Bu-C14, demonstrates a guest-adaptive hosting capability of TNAs for the first time. When binding fullerene guests, the cavity of 6L-Bu-C14 became more circular to better accommodate spherical fullerene molecules. The host-guest interaction is thoroughly studied by X-ray crystallography, theoretical calculations, fluorescence titration, and nuclear magnetic resonance (NMR) titration experiments. 6L-Bu-C14 shows stronger binding with C70 than with C60 due to the better convex-concave π-π interaction. P and M enantiomers of all tethered aromatic lactams show distinct and persistent chiroptical properties and demonstrate the potential of chiral TNAs as circularly polarized luminescence (CPL) emitters.

Scaling and merging macromolecular diffuse scattering with mdx2.

Diffuse scattering is a promising method to gain additional insight into protein dynamics from macromolecular crystallography experiments. Bragg intensities yield the average electron density, while the diffuse scattering can be processed to obtain a three-dimensional reciprocal-space map that is further analyzed todetermine correlated motion. To make diffuse scattering techniques more accessible, software for data processing called mdx2 has been created that is both convenient to use and simple to extend and modify. mdx2 is written in Python, and it interfaces with DIALS to implement self-contained data-reduction workflows. Data are stored in NeXus format for software interchange and convenient visualization. mdx2 can be run on the command line or imported as a package, for instance to encapsulate a complete workflow in a Jupyter notebook for reproducible computing and education. Here, mdx2 version 1.0 is described, a new release incorporating state-of-the-art techniques for data reduction. The implementation of a complete multi-crystal scaling and merging workflow is described, and the methods are tested using a high-redundancy data set from cubic insulin. It is shown that redundancy can be leveraged during scaling to correct systematic errors and obtain accurate and reproducible measurements of weak diffuse signals.

Accurate Characterization of Binding Kinetics and Allosteric Mechanisms for the HSP90 Chaperone Inhibitors Using AI-Augmented Integrative Biophysical Studies

Accurate Characterization of Binding Kinetics and Allosteric Mechanisms for the HSP90 Chaperone Inhibitors Using AI-Augmented Integrative Biophysical Studies

Robust image descriptor for machine learning based data reduction in serial crystallography.

Serial crystallography experiments at synchrotron and X-ray free-electron laser (XFEL) sources are producing crystallographic data sets of ever-increasing volume. While these experiments have large data sets and high-frame-rate detectors (around 3520 frames per second), only a small percentage of the data are useful for downstream analysis. Thus, an efficient and real-time data classification pipeline is essential to differentiate reliably between useful and non-useful images, typically known as 'hit' and 'miss', respectively, and keep only hit images on disk for further analysis such as peak finding and indexing. While feature-point extraction is a key component of modern approaches to image classification, existing approaches require computationally expensive patch preprocessing to handle perspective distortion. This paper proposes a pipeline to categorize the data, consisting of a real-time feature extraction algorithm called modified and parallelized FAST (MP-FAST), an image descriptor and a machine learning classifier. For parallelizing the primary operations of the proposed pipeline, central processing units, graphics processing units and field-programmable gate arrays are implemented and their performances compared. Finally, MP-FAST-based image classification is evaluated using a multi-layer perceptron on various data sets, including both synthetic and experimental data. This approach demonstrates superior performance compared with other feature extractors and classifiers.

An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy.

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins.

Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations.

The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependent oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long-range propagation of cooperative effects across the hNQO1 structure. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether, these results pave the way for future time-resolved studies, both at x-ray free-electron lasers and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half-reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1.

Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE.

X-ray Free Electron Lasers (XFELs) allow the collection of high-quality serial femtosecond crystallography data. The next generation of megahertz superconducting FELs promises to drastically reduce data collection times, enabling the capture of more structures with higher signal-to-noise ratios and facilitating more complex experiments. Currently, gas dynamic virtual nozzles (GDVNs) stand as the sole delivery method capable of best utilizing the repetition rate of megahertz sources for crystallography. However, their substantial sample consumption renders their use impractical for many protein targets in serial crystallography experiments. Here, we present a novel application of a droplet-on-demand injection method, which allowed operation at 47 kHz at the European XFEL (EuXFEL) by tailoring a multi-droplet injection scheme for each macro-pulse. We demonstrate a collection rate of 150 000 indexed patterns per hour. We show that the performance and effective data collection rate are comparable to GDVN, with a sample consumption reduction of two orders of magnitude. We present lysozyme crystallographic data using the Large Pixel Detector at the femtosecond x-ray experiment endstation. Significant improvement of the crystallographic statistics was made by correcting for a systematic drift of the photon energy in the EuXFEL macro-pulse train, which was characterized from indexing the individual frames in the pulse train. This is the highest resolution protein structure collected and reported at the EuXFEL at 1.38 Å resolution.

Crystallographic Data Collection Using a Multilayer Monochromator on an Undulator Beamline at the Shanghai Synchrotron Radiation Facility

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods.

The Pixel Anomaly Detection Tool: a user-friendly GUI for classifying detector frames using machine-learning approaches.

Data collection at X-ray free electron lasers has particular experimental challenges, such as continuous sample delivery or the use of novel ultrafast high-dynamic-range gain-switching X-ray detectors. This can result in a multitude of data artefacts, which can be detrimental to accurately determining structure-factor amplitudes for serial crystallography or single-particle imaging experiments. Here, a new data-classification tool is reported that offers a variety of machine-learning algorithms to sort data trained either on manual data sorting by the user or by profile fitting the intensity distribution on the detector based on the experiment. This is integrated into an easy-to-use graphical user interface, specifically designed to support the detectors, file formats and software available at most X-ray free electron laser facilities. The highly modular design makes the tool easily expandable to comply with other X-ray sources and detectors, and the supervised learning approach enables even the novice user to sort data containing unwanted artefacts or perform routine data-analysis tasks such as hit finding during an experiment, without needing to write code.

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.

Application of Micro-Tubing Reeling System to Serial Femtosecond Crystallography

Microcrystal delivery instruments are pivotal to performing serial femtosecond crystallography experiments at the XFEL facilities. We present a novel sample delivery technique based on a micro-tubing reeling system (MRS). Despite the tiny size of the micro-tubing, the MRS device has the advantage of operating without real-time position adjustment of the tube to match with the XFEL pulses. Moreover, the applicable repetition rate is more flexible than the previously reported chip-based one-dimensional fixed target system.

Structural Basis for p19 Targeting by Anti–IL-23 Biologics: Correlations with Short- and Long-Term Efficacy in Psoriasis

IL-23 is central to psoriasis pathogenesis. Biologics targeting IL-23 are important therapies against psoriasis. IL-23 inhibitors risankizumab, tildrakizumab, and guselkumab bind the IL-23 p19 subunit, whereas ustekinumab binds p40; however, the structural composition of the IL-23-binding epitopes and how these molecular properties relate to clinical efficacy are not known. Utilizing epitope data derived from hydrogen-deuterium exchange or crystallographic experiments, we mapped inhibitor epitope locations, hydrophobicity, and surface charge onto the IL-23 surface. Molecular properties of each inhibitor epitope, including solvent-accessible surface area, were correlated to binding affinity, kinetic values, and clinical efficacy scores for plaque psoriasis through linear regression analysis. Each IL-23 inhibitor binds an epitope with a unique size, composition, and location except for a 10-residue overlap region outside of the IL-23 receptor epitope. We observed strong correlations between epitope surface area and KD and koff but not kon. Epitope surface area, KD, and koff were further associated with short-term (10-16 weeks) and long-term (44-60 weeks) clinical efficacy according to PASI-90 responses, with risankizumab demonstrating highest efficacy among IL-23 biologics. In contrast, kon, epitope hydrophobicity, polarity, and charge content did not correlate with efficacy. These data exemplify how molecular principles of medications within a therapeutic class can explain their differential clinical responses.

Light-induced Trpin/Metout Switching During BLUF Domain Activation in ATP-bound Photoactivatable Adenylate Cyclase OaPAC

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Polarisation effects on the H-bond acceptor properties of sulfonamides.

The strengths of H-bonding interactions in networks are affected by cooperativity between the interacting sites. Compounds with an intramolecular H-bond between a sulfonamide NH group and pyridine nitrogen were used to measure the magnitude of cooperative effects on intermolecular H-bonding interactions with the sulfonamide oxygen. X-ray crystallography and 1H NMR experiments confirm the presence of the intramolecular H-bond and show that it is maintained in the 1 : 1 complex formed with perfluoro-tert-butanol (PFTB) in n-octane solution. Association constants for formation of 1 : 1 complexes with PFTB were determined using UV/Vis absorption titrations for a series of compounds equipped with different pyridine groups. Substituents on the pyridine were used to tune the strength of the intramolecular H-bond and investigate the effects on the strength of the intermolecular H-bond. Electron-donating groups on the pyridine that increase the strength of the intramolecular H-bond were found to increase in the strength of the intermolecular interaction with PFTB. The results were used to determine the H-bond acceptor parameters, β, for the sulfonamide oxygen group, and the values show a linear relationship with the value of β for the pyridine nitrogen. The slope of this relationship corresponds to the cooperativity parameter, κ, which is +0.16. The positive cooperativity observed in H-bonded sulfonamides is comparable to the value measured previously for the amide group (κ = +0.20).

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers.

Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology.

Impact of Low-Temperature Water Exposure and Removal on Zeolite HY.

Aqueous-phase postsynthetic modifications of the industrially important Y-type zeolite are commonly used to change overall acid site concentrations, introduce stabilizing rare-earth cations, impart bifunctional character through metal cation exchange, and tailor the distribution of Brønsted and Lewis acid sites. Zeolite Y is known to undergo framework degradation in the presence of both vapor- and liquid-phase water at temperatures exceeding 100 °C, and rare-earth exchanged and stabilized HY catalysts are commonly used for fluidized catalytic cracking due to their increased hydrothermal resilience. Here, using detailed spectroscopy, crystallography, and flow-reactor experiments, we reveal unexpected decreases in Brønsted acid site (BAS) density for zeolite HY following exposure even to room-temperature liquid water. These data indicate that aqueous-phase ion-exchange procedures commonly used to modify zeolite Y are impacted by the liquid water and its removal, even when fractional heating rates and inert conditions much less severe than standard practice are used for catalyst dehydration. X-ray diffraction, thermogravimetric, and spectroscopic analyses reveal that the majority of framework degradation occurs during the removal of a strongly bound water fraction in HY, which does not form when NH4Y is immersed in liquid water and which leads to reduced acidity in HY even when dehydration conditions much milder than those typically practiced are employed. Na+-exchanged HY prepared via room-temperature aqueous dissolution demonstrates that Brønsted acid sites are lost in excess of the theoretical maximum that is possible from sodium titration. The structural impact of low-temperature aqueous-phase ion-exchange methods complicates the interpretation of subsequent data and likely explains the wide variation in reported acid site concentrations and catalytic activity of HY zeolites with high-Al content.

Synthesis and cytotoxicity evaluation of retro-inverso analogue of antitumor bicyclic hexapeptide RA-VII

A retro-inverso analogue of antitumor bicyclic hexapeptide RA-VII was designed and synthesized. A cycloisodityrosine unit was synthesized by the intramolecular diphenyl ether formation of Boc-3-borono-d-Tyr(Me)-d-Tyr-OMe mediated by copper(II) acetate and 4-(dimethylamino)pyridine. After the N-methylation, Boc-Ala-OH and the tripeptide segment were sequentially coupled to the N-terminus of the cycloisodityrosine unit, affording a hexapeptide. After removal of the C- and N-terminal protecting groups, the hexapeptide was subjected to macrocyclization using diphenylphosphoryl azide and NaHCO3 in N,N-dimethylformamide to afford a bicyclic hexapeptide analogue. X-ray crystallography and NMR experiments showed that the analogue had different structural features from RA-VII, which were responsible for the lack of cytotoxic activity of the analogue.

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.