Time-resolved Crystallography Research Articles

Crystal structure of a bacterial photoactivated adenylate cyclase determined by serial femtosecond and serial synchrotron crystallography.

OaPAC is a recently discovered blue-light-using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report crystal structures of the enzyme in the absence of its natural substrate determined from room-temperature serial crystallography data collected at both an X-ray free-electron laser and a synchrotron, and we compare these structures with cryo-macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6W mutation in the BLUF domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side chain of the critical Gln48 residue. These studies pave the way for picosecond-millisecond time-resolved serial crystallography experiments at X-ray free-electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well studied picosecond-millisecond spectroscopic intermediates.

ResiDEM: Analytical Tool for Isomorphous Difference Electron Density Maps Utilizing Dynamic Residue Identification via Density Clustering.

Time-resolved serial femtosecond crystallography (TR-SFX) of biological molecules captures the time-evolved dynamics of the residual motions across crystal structures, enabling the visualization of structural changes in response to chemical and physical stimuli to elucidate the relationship between the structure and function of the system under study. However, interpretations of residual motions can be complex to deconvolute because of various factors such as the system's size, temporal and spatial complexity, and allosteric behavior away from active sites. Relying solely on electron density map visualization can also pose a challenge in differentiating between useful and irrelevant data. In order to accurately identify residues and determine their respective contributions to the reaction dynamics, new tools are needed. We developed a new tool, ResiDEM, which employs a clustering-based approach to group difference electron densities and associate them with proximal residues. It can identify and rank residues with significant motions. Network representation can be used to delineate the interrelations between the residues in motion. With these features, ResiDEM helps to interpret residual motions in TR-SFX data, identify key residues, and elucidate their roles in dynamic processes.

Single pulse data collection with an X-ray chopper at in situ room temperature Laue crystallography beamline BL03HB

in situ room temperature Laue crystallography has gained increasing attention. This method is suitable for time-resolved Laue crystallography experiments to get crystal structure with only several single-frame data collections. To improve the diffraction efficiency of protein crystals at room temperature, a simple X-ray chopper combined with a fast shutter has been successfully developed and tested at in situ room temperature Laue crystallography beamline BL03HB of the Shanghai Synchrotron Radiation Facility (SSRF). This chopper is built with two aluminum blades. There are two windows on both blades, as well as an internal lead block. The width of windows for transmission of beam can be flexibly adjusted by a steel block coated with lead. The chopping frequency is controlled independently. The device allows chopping frequencies ranging from 0 to 10 Hz, and exposure times down to 100 μs can be obtained for our Laue diffraction experiment with the combination of a fast shutter. Moreover, a single pulse data collection strategy has been developed at BL03HB. The performance of our single pulse data collection method with the designed chopper for the improvement of data quality has been analyzed. Results show that the quality of diffraction data obtained from the single pulse data collection method is significantly improved, and the developed chopper is very suitable for room temperature Laue crystallography experiments at the synchrotron beamline with super bending magnet as light source.

The structure of Chlamydomonas LOV1 as revealed by time-resolved serial synchrotron crystallography.

The photo-reaction of the LOV1 domain of the Chlamydomonas reinhardtii phototropin is investigated by room-temperature time-resolved serial crystallography. A covalent adduct forms between the C4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined 23 years ago from the maidenhair fern Phy3.

Innovative Strategies in X-ray Crystallography for Exploring Structural Dynamics and Reaction Mechanisms in Metabolic Disorders.

Enzymes are crucial in metabolic processes, and their dysfunction can lead to severe metabolic disorders. Structural biology, particularly X-ray crystallography, has advanced our understanding of these diseases by providing 3D structures of pathological enzymes. However, traditional X-ray crystallography faces limitations, such as difficulties in obtaining suitable protein crystals and studying protein dynamics. X-ray free-electron lasers (XFELs) have revolutionized this field with their bright and brief X-ray pulses, providing high-resolution structures of radiation-sensitive and hard-to-crystallize proteins. XFELs also enable the study of protein dynamics through room temperature structures and time-resolved serial femtosecond crystallography, offering comprehensive insights into the molecular mechanisms of metabolic diseases. Understanding these dynamics is vital for developing effective therapies. This review highlights the contributions of protein dynamics studies using XFELs and synchrotrons to metabolic disorder research and their application in designing better therapies. It also discusses G protein-coupled receptors (GPCRs), which, though not enzymes, play key roles in regulating physiological systems and are implicated in many metabolic disorders.

Time-Resolved Serial Femtosecond Crystallography of Cytochrome c Oxidase by use of a photolabile oxygen cage and novel insights of the catalytic site from high level multiconfigurational ab initio calculations

Time-Resolved Serial Femtosecond Crystallography of Cytochrome c Oxidase by use of a photolabile oxygen cage and novel insights of the catalytic site from high level multiconfigurational ab initio calculations

Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo.

Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.

Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo

Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.

Time-resolved serial femtosecond crystallography for investigating structural dynamics of chemical systems.

Time-resolved serial femtosecond crystallography (TR-SFX) has emerged as a crucial tool for studying the structural dynamics of proteins. In principle, TR-SFX has the potential to be a powerful tool not only for studying proteins but also for investigating chemical reactions. However, non-protein systems generally face challenges in indexing due to sparse Bragg spots and encounter difficulties in effectively exciting target molecules. Nevertheless, successful TR-SFX studies on chemical systems have been recently reported in a few instances, boding well for the application of TR-SFX to study chemical reactions in the future. In this context, we review the static SFX and TR-SFX studies conducted on chemical systems reported to date and suggest prospects for future research directions.

XFEL Beamline Optical Instrumentation for Ultrafast Science.

Free electron lasers operating in the soft and hard X-ray regime provide capabilities for ultrafast science in many areas, including X-ray spectroscopy, diffractive imaging, solution and material scattering, and X-ray crystallography. Ultrafast time-resolved applications in the picosecond, femtosecond, and attosecond regimes are often possible using single-shot experimental configurations. Aside from X-ray pump and X-ray probe measurements, all other types of ultrafast experiments require the synchronized operation of pulsed laser excitation for resonant or nonresonant pumping. This Perspective focuses on the opportunities for the optical control of structural dynamics by applying techniques from nonlinear spectroscopy to ultrafast X-ray experiments. This typically requires the synthesis of two or more optical pulses with full control of pulse and interpulse parameters. To this end, full characterization of the femtosecond optical pulses is also highly desirable. It has recently been shown that two-color and two-pulse femtosecond excitation of fluorescent protein crystals allowed a Tannor-Rice coherent control experiment, performed under characterized conditions. Pulse shaping and the ability to synthesize multicolor and multipulse conditions are highly desirable and would enable XFEL facilities to offer capabilities for structural dynamics. This Perspective will give a summary of examples of the types of experiments that could be achieved, and it will additionally summarize the laser, pulse shaping, and characterization that would be recommended as standard equipment for time-resolved XFEL beamlines, with an emphasis on ultrafast time-resolved serial femtosecond crystallography.

Scaling and Merging Time-Resolved Laue Data with Variational Inference.

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.

Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family.

The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.

A snapshot love story: what serial crystallography has done and will do for us.

Serial crystallography, born from groundbreaking experiments at the Linac Coherent Light Source in 2009, has evolved into a pivotal technique in structural biology. Initially pioneered at X-ray free-electron laser facilities, it has now expanded to synchrotron-radiation facilities globally, with dedicated experimental stations enhancing its accessibility. This review gives an overview of current developments in serial crystallography, emphasizing recent results in time-resolved crystallography, and discussing challenges and shortcomings.

Relevance of G protein-coupled receptor (GPCR) dynamics for receptor activation, signalling bias and allosteric modulation.

G protein-coupled receptors (GPCRs) are one of the major drug targets. In recent years, computational drug design for GPCRs has mainly focused on static structures obtained through X-ray crystallography, cryogenic electron microscopy (cryo-EM) or in silico modelling as a starting point for virtual screening campaigns. However, GPCRs are highly flexible entities with the ability to adopt different conformational states that elicit different physiological responses. Including this knowledge in the drug discovery pipeline can help to tailor novel conformation-specific drugs with an improved therapeutic profile. In this review, we outline our current knowledge about GPCR dynamics that is relevant for receptor activation, signalling bias and allosteric modulation. Ultimately, we highlight new technological implementations such as time-resolved X-ray crystallography and cryo-EM as well as computational algorithms that can contribute to a more comprehensive understanding of receptor dynamics and its relevance for GPCR functionality.

Time-resolved crystallography of boric acid binding to the active site serine of the β-lactamase CTX-M-14 and subsequent 1,2-diol esterification

The emergence and spread of antibiotic resistance represent a growing threat to public health. Of particular concern is the appearance of β-lactamases, which are capable to hydrolyze and inactivate the most important class of antibiotics, the β-lactams. Effective β-lactamase inhibitors and mechanistic insights into their action are central in overcoming this type of resistance, and in this context boronate-based β-lactamase inhibitors were just recently approved to treat multidrug-resistant bacteria. Using boric acid as a simplified inhibitor model, time-resolved serial crystallography was employed to obtain mechanistic insights into binding to the active site serine of β-lactamase CTX-M-14, identifying a reaction time frame of 80–100 ms. In a next step, the subsequent 1,2-diol boric ester formation with glycerol in the active site was monitored proceeding in a time frame of 100–150 ms. Furthermore, the displacement of the crucial anion in the active site of the β-lactamase was verified as an essential part of the binding mechanism of substrates and inhibitors. In total, 22 datasets of β-lactamase intermediate complexes with high spatial resolution of 1.40–2.04 Å and high temporal resolution range of 50–10,000 ms were obtained, allowing a detailed analysis of the studied processes. Mechanistic details captured here contribute to the understanding of molecular processes and their time frames in enzymatic reactions. Moreover, we could demonstrate that time-resolved crystallography can serve as an additional tool for identifying and investigating enzymatic reactions.

Structural Dynamics of Metalloproteins and Redox Enzymology with Mix-and-Inject Time-Resolved Serial Femtosecond Crystallography

Structural Dynamics of Metalloproteins and Redox Enzymology with Mix-and-Inject Time-Resolved Serial Femtosecond Crystallography

Appraising protein conformational changes by resampling time-resolved serial x-ray crystallography data.

With the development of serial crystallography at both x-ray free electron laser and synchrotron radiation sources, time-resolved x-ray crystallography is increasingly being applied to study conformational changes in macromolecules. A successful time-resolved serial crystallography study requires the growth of microcrystals, a mechanism for synchronized and homogeneous excitation of the reaction of interest within microcrystals, and tools for structural interpretation. Here, we utilize time-resolved serial femtosecond crystallography data collected from microcrystals of bacteriorhodopsin to compare results from partial occupancy structural refinement and refinement against extrapolated data. We illustrate the domain wherein the amplitude of refined conformational changes is inversely proportional to the activated state occupancy. We illustrate how resampling strategies allow coordinate uncertainty to be estimated and demonstrate that these two approaches to structural refinement agree within coordinate errors. We illustrate how singular value decomposition of a set of difference Fourier electron density maps calculated from resampled data can minimize phase bias in these maps, and we quantify residual densities for transient water molecules by analyzing difference Fourier and Polder omit maps from resampled data. We suggest that these tools may assist others in judging the confidence with which observed electron density differences may be interpreted as functionally important conformational changes.

Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

Room-temperature serial synchrotron crystallography structure of Spinacia oleracea RuBisCO.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the enzyme responsible for the first step of carbon dioxide (CO2) fixation in plants, which proceeds via the carboxylation of ribulose 1,5-biphosphate. Because of the enormous importance of this reaction in agriculture and the environment, there is considerable interest in the mechanism of fixation of CO2 by RuBisCO. Here, a serial synchrotron crystallography structure of spinach RuBisCO is reported at2.3 Å resolution. This structure is consistent with earlier single-crystal X-ray structures of this enzyme and the results are a good starting point for a further push towards time-resolved serial synchrotron crystallography in order to better understand the mechanism of the reaction.