ABSTRACT
Dental implant surface properties such as roughness, wettability, and porosity ensure cell interaction and tissue integration. The clinical performance of dental implants depends on the crystallographic texture and protein and cell bonds to the substrates, where grain size, orientation, and inclination are parameters responsible for favoring osteoblast adhesion and limiting bacterial adhesion. The lack of consensus on the best crystallographic plan for cell adhesion prompted this systematic review, which aims to answer the following question: "What is the influence of the crystallographic plane on titanium surfaces on cell adhesion?" by evaluating the literature on the crystallographic characteristics of titanium and how these dictate topographical parameters and influence the cell adhesion of devices made from this material. It followed the Preferred Reporting Standards for Systematic Reviews and Meta-Analyses (PRISMA 2020) registered with the Open Science Framework (OSF) (osf.io/xq6kv). The search strategy was based on the PICOS method. It chose in vitro articles that analyzed crystallographic structure correlated with cell adhesion and investigated the microstructure and its effects on cell culture, different crystal orientation distributions, and the influence of crystallinity. The search strategies were applied to the different electronic databases: PubMed, Scopus, Science Direct, Embase, and Google Scholar, and the articles found were attached to the Rayyan digital platform and assessed blindly. The Joanna Bringgs Institute (JBI) tool assessed the risk of bias. A total of 248 articles were found. After removing duplicates, 192 were analyzed by title and abstract. Of these, 18 were selected for detailed reading in their entirety, 9 of which met the eligibility criteria. The included studies presented a low risk of bias. The role of the crystallographic orientation of the exposed faces in a multicrystalline material is little discussed in the scientific literature and its impact is recognized as dictating the topographical characteristics of the material that facilitate cell adhesion.
Subject(s)
Cell Adhesion , Titanium , Titanium/chemistry , Humans , Surface Properties , Dental Implants , Crystallography , Animals , Osteoblasts/metabolism , Osteoblasts/cytologyABSTRACT
A series of events underscoring the significant advancements in micro-crystallization and in vivo crystallography were held during the 26th IUCr Congress in Melbourne, positioning microcrystallography as a pivotal field within structural biology. Through collaborative discussions and the sharing of innovative methodologies, these sessions outlined frontier approaches in macromolecular crystallography. This review provides an overview of this rapidly moving field in light of the rich dialogues and forward-thinking proposals explored during the congress workshop and microsymposium. These advances in microcrystallography shed light on the potential to reshape current research paradigms and enhance our comprehension of biological mechanisms at the molecular scale.
Subject(s)
Crystallization , Crystallography, X-Ray/methods , Crystallography/methods , Macromolecular Substances/chemistryABSTRACT
Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today - conventional X-ray crystallography - is that it only provides structural information about the protein complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room temperature (RT). Here, we explore the use of serial crystallography to determine the structures of the pharmaceutical target, soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of RT ligand-bound structures of our target protein. From a comparison between the RT structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in the ligand-binding mode and observe that flexible loops are better resolved at RT. Finally, we discuss the current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.
Subject(s)
Drug Discovery , Epoxide Hydrolases , Drug Discovery/methods , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Crystallography, X-Ray/methods , Protein Conformation , Ligands , Humans , Temperature , Models, Molecular , Crystallography/methods , CrystallizationABSTRACT
Caudofoveata are molluscs that protect their vermiform body with a scleritome, a mosaic of unconnected blade/lanceolate-shaped aragonite sclerites. For the species Falcidens gutturosus and Scutopus ventrolineatus we studied the crystallographic constitution and crystal orientation texture of the sclerites and the scleritome with electron-backscatter-diffraction (EBSD), laser-confocal-microscopy (LCM) and field-emission electron microscopy (FE-SEM) imaging. Each sclerite is an aragonite single crystal that is completely enveloped by an organic sheath. Adjacent sclerites overlap laterally and vertically are, however, not connected to each other. Sclerites are thickened in their central portion, relative to their periphery. Thickening increases also from sclerite tip towards its base. Accordingly, cross-sections through a sclerite are straight at its tip, curved and bent towards the sclerite base. Irrespective of curved sclerite morphologies, the aragonite lattice within the sclerite is coherent. Sclerite aragonite is not twinned. For each sclerite the crystallographic c-axis is parallel to the morphological long axis of the sclerite, the a-axis is perpendicular to its width and the b-axis is within the width of the sclerite. The single-crystalinity of the sclerites and their mode of organization in the scleritome is outstanding. Sclerite and aragonite arrangement in the scleritome is not given by a specific crystal growth mode, it is inherent to the secreting cells. We discuss that morphological characteristics of the sclerites and crystallographic preferred orientation (texture) of sclerite aragonite is not the result of competitive growth selection. It is generated by the templating effect of the organic substance of the secreting cells and associated extracellular biopolymers.
Subject(s)
Animal Shells , Calcium Carbonate , Mollusca , Animals , Animal Shells/chemistry , Animal Shells/ultrastructure , Mollusca/chemistry , Calcium Carbonate/chemistry , Crystallography , Microscopy, Electron, ScanningABSTRACT
OBJECTIVES: This study aimed to determine the crystalline phase composition of 3-6 mol% yttria-stabilized zirconia (3-6YSZ), specifically investigating the presence of tetragonal (t), cubic (c), and/or additional yttria-rich tetragonal (t') phase. METHODS: Laboratory-fabricated specimens comprising 3-5YSZ, resembling translucent dental zirconia ceramics (TZ specimens), and a blend of 3YSZ and 8YSZ, representing a c-phase reference, were prepared. Additionally, 25 dental zirconia products stabilized with 3-6 mol% yttria were analyzed. Whole X-ray diffraction (XRD) patterns were obtained for Rietveld analysis, complemented by fine scanning in the 2θ region from 72º to 76º for qualitative phase analysis. Moreover, yttria concentrations in each specimen were determined using X-ray fluorescence (XRF) spectroscopy. RESULTS: In the 2θ region from 72º to 76º, TZ and dental zirconia product specimens displayed four peaks attributed to t- and t'-phases, but the c-phase peak was absent. Rietveld analysis of the whole XRD patterns, utilizing a t-t' model, demonstrated the t-phase fraction ranging from 86 mass% in 3YSZ to 11 mass% in 6YSZ. Rietveld analysis appeared reliable, as the yttria contents calculated based on lattice parameters aligned well with those measured by XRF. This study established that dental 3-6YSZ consisted of yttria-lean t- and yttria-rich t'-phases. SIGNIFICANCE: The present study enhances understanding of the crystalline structure of dental zirconia ceramics. Future crystallographic analyses of these ceramics should consider the presence of t- and t'-phases.
Subject(s)
Ceramics , Materials Testing , X-Ray Diffraction , Yttrium , Zirconium , Zirconium/chemistry , Yttrium/chemistry , Ceramics/chemistry , Surface Properties , Spectrometry, X-Ray Emission , Crystallography , Dental Materials/chemistryABSTRACT
Aplacophoran molluscs are shell-less and have a worm-like body which is covered by biomineralized sclerites. We investigated sclerite crystallography and the sclerite mosaic of the Solenogastres species Dorymenia sarsii, Anamenia gorgonophila, and Simrothiella margaritacea with electron-backscattered-diffraction (EBSD), laser-confocal-microscopy and FE-SEM imaging. The soft tissue of the molluscs is covered by spicule-shaped, aragonitic sclerites. These are sub-parallel to the soft body of the organism. We find, for all three species, that individual sclerites are untwinned aragonite single crystals. For individual sclerites, aragonite c-axis is parallel to the morphological, long axis of the sclerite. Aragonite a- and b-axes are perpendicular to sclerite aragonite c-axis. For the scleritomes of the investigated species we find different sclerite and aragonite crystal arrangement patterns. For the A. gorgonophila scleritome, sclerite assembly is disordered such that sclerites with their morphological, long axis (always the aragonite c-axis) are pointing in many different directions, being, more or less, tangential to cuticle surface. For D. sarsii, the sclerite axes (equal to aragonite c-axes) show a stronger tendency to parallel arrangement, while for S. margaritacea, sclerite and aragonite organization is strongly structured into sequential rows of orthogonally alternating sclerite directions. The different arrangements are well reflected in the structured orientational distributions of aragonite a-, b-, c-axes across the EBSD-mapped parts of the scleritomes. We discuss that morphological and crystallographic preferred orientation (texture) is not generated by competitive growth selection (the crystals are not in contact), but is determined by templating on organic matter of the sclerite-secreting epithelial cells and associated papillae.
Subject(s)
Mollusca , Animals , Mollusca/chemistry , Calcium Carbonate/chemistry , Crystallography/methods , Biomineralization , Animal Shells/chemistry , Microscopy, Electron, ScanningABSTRACT
Deep-learning methods have revolutionized protein structure prediction and design but are presently limited to protein-only systems. We describe RoseTTAFold All-Atom (RFAA), which combines a residue-based representation of amino acids and DNA bases with an atomic representation of all other groups to model assemblies that contain proteins, nucleic acids, small molecules, metals, and covalent modifications, given their sequences and chemical structures. By fine-tuning on denoising tasks, we developed RFdiffusion All-Atom (RFdiffusionAA), which builds protein structures around small molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we designed and experimentally validated, through crystallography and binding measurements, proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and the light-harvesting molecule bilin.
Subject(s)
Deep Learning , Protein Engineering , Proteins , Amino Acids/chemistry , Crystallography , DNA/chemistry , Models, Molecular , Proteins/chemistry , Protein Engineering/methodsABSTRACT
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.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Crystallography , Temperature , NAD , Antineoplastic Agents/chemistry , Flavins , Crystallography, X-Ray , NAD(P)H Dehydrogenase (Quinone)ABSTRACT
Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3â µm crystals of lysozyme and 2â µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2â ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.
Subject(s)
Arabidopsis , Microfluidics , Crystallography , Cognition , ConvectionABSTRACT
Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.
Subject(s)
5-Methylcytosine , DNA , Crystallization , Catalysis , CrystallographyABSTRACT
High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.
Subject(s)
Artifacts , Lasers , Myoglobin , Crystallography/instrumentation , Crystallography/methods , Electrons , Myoglobin/chemistry , Myoglobin/metabolism , Myoglobin/radiation effects , Photons , Protein Conformation/radiation effects , Quantum Theory , X-RaysABSTRACT
Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD+. The multi-step reaction mechanism involves an exchange of bound NAD+ and large conformational changes by a 50-residue subdomain. The enzymatic reaction can be run in both forward and reverse directions in solution and is catalytically active in the crystal for multiple reaction steps. Initially, the enzyme was found to be inactive in the crystal starting with bound mevalonate, CoA, and NAD+. To observe the reaction from this direction, we examined the effects of crystallization buffer constituents and pH on enzyme turnover, discovering a strong inhibition in the crystallization buffer and a controllable increase in enzyme turnover as a function of pH. The inhibition is dependent on ionic concentration of the crystallization precipitant ammonium sulfate but independent of its ionic composition. Crystallographic studies show that the observed inhibition only affects the oxidation of mevalonate but not the subsequent reactions of the intermediate mevaldehyde. Calculations of the pKa values for the enzyme active site residues suggest that the effect of pH on turnover is due to the changing protonation state of His381. We have now exploited the changes in ionic inhibition in combination with the pH-dependent increase in turnover as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about its intermediate states along the reaction pathway.
Subject(s)
Hydroxymethylglutaryl CoA Reductases , NAD , Hydroxymethylglutaryl CoA Reductases/chemistry , Hydroxymethylglutaryl CoA Reductases/metabolism , NAD/metabolism , Crystallography , Mevalonic Acid/metabolism , Hydrogen-Ion Concentration , KineticsABSTRACT
Serial crystallography (SX) has become an established technique for protein structure determination, especially when dealing with small or radiation-sensitive crystals and investigating fast or irreversible protein dynamics. The advent of newly developed multi-megapixel X-ray area detectors, capable of capturing over 1000 images per second, has brought about substantial benefits. However, this advancement also entails a notable increase in the volume of collected data. Today, up to 2â PB of data per experiment could be easily obtained under efficient operating conditions. The combined costs associated with storing data from multiple experiments provide a compelling incentive to develop strategies that effectively reduce the amount of data stored on disk while maintaining the quality of scientific outcomes. Lossless data-compression methods are designed to preserve the information content of the data but often struggle to achieve a high compression ratio when applied to experimental data that contain noise. Conversely, lossy compression methods offer the potential to greatly reduce the data volume. Nonetheless, it is vital to thoroughly assess the impact of data quality and scientific outcomes when employing lossy compression, as it inherently involves discarding information. The evaluation of lossy compression effects on data requires proper data quality metrics. In our research, we assess various approaches for both lossless and lossy compression techniques applied to SX data, and equally importantly, we describe metrics suitable for evaluating SX data quality.
Subject(s)
Algorithms , Data Compression , Crystallography , Data Compression/methods , Tomography, X-Ray ComputedABSTRACT
ASPP2 and iASPP bind to p53 through their conserved ANK-SH3 domains to respectively promote and inhibit p53-dependent cell apoptosis. While crystallography has indicated that these two proteins employ distinct surfaces of their ANK-SH3 domains to bind to p53, solution NMR data has suggested similar surfaces. In this study, we employed multi-scale molecular dynamics (MD) simulations combined with free energy calculations to reconcile the discrepancy in the binding modes. We demonstrated that the binding mode based solely on a single crystal structure does not enable iASPP's RT loop to engage with p53's C-terminal linker-a verified interaction. Instead, an ensemble of simulated iASPP-p53 complexes facilitates this interaction. We showed that the ensemble-average inter-protein contacting residues and NMR-detected interfacial residues qualitatively overlap on ASPP proteins, and the ensemble-average binding free energies better match experimental KD values compared to single crystallgarphy-determined binding mode. For iASPP, the sampled ensemble complexes can be grouped into two classes, resembling the binding modes determined by crystallography and solution NMR. We thus propose that crystal packing shifts the equilibrium of binding modes towards the crystallography-determined one. Lastly, we showed that the ensemble binding complexes are sensitive to p53's intrinsically disordered regions (IDRs), attesting to experimental observations that these IDRs contribute to biological functions. Our results provide a dynamic and ensemble perspective for scrutinizing these important cancer-related protein-protein interactions (PPIs).
Subject(s)
Apoptosis Regulatory Proteins , Tumor Suppressor Protein p53 , Apoptosis Regulatory Proteins/chemistry , Tumor Suppressor Protein p53/chemistry , Crystallography , Protein Binding , ApoptosisABSTRACT
The development of NMR crystallography methods requires a reliable database of chemical shifts measured for systems with known crystal structure. We measured and assigned carbon and hydrogen chemical shifts of twenty solid natural amino acids of known polymorphic structure, meticulously determined using powder X-ray diffraction. We then correlated the experimental data with DFT-calculated isotropic shieldings. The small size of the unit cell of most amino acids allowed for advanced computations using various families of DFT functionals, including generalized gradient approximation (GGA), meta-GGA and hybrid DFT functionals. We tested several combinations of functionals for geometry optimizations and NMR calculations. For carbon shieldings, the widely used GGA functional PBE performed very well, although an improvement could be achieved by adding shielding corrections calculated for isolated molecules using a hybrid functional. For hydrogen nuclei, we observed the best performance for NMR calculations carried out with structures optimized at the hybrid DFT level. The high fidelity of the calculations made it possible to assign additional signals that could not be assigned based on experiments alone, for example signals of two non-equivalent molecules in the unit cell of some of the amino acids.
Subject(s)
Amino Acids , Carbon , Crystallography , Magnetic Resonance Spectroscopy/methods , HydrogenABSTRACT
Due to their structural diversities and prevalent biological activities, natural products (NPs) are momentous resources for drug discovery. Although NPs have a wide range of biological activities, many exhibit structural complexity that leads to synthetic difficulties, which combines with inefficient biological activity, toxicity, and unfavorable pharmacokinetic characteristics and ultimately imparts poor safety and efficacy outcomes. Progress in crystallization and computational techniques allow crystallography to have a seasonable influences on drug discovery. By co-crystallizing with proteins, therapeutic targets of NPs in specific diseases can be identified. By analyzing the co-crystal information, the structure-activity relationships (SARs) of NPs targeting specific proteins can be grasped. Under the guidance of co-crystal information, directional structural modification and simplification are powerful strategies for overcoming limitations of NPs, improving the success rate of NP-based drug discovery, and obtaining NP-based drugs with high selectivity, low toxicity and favorable pharmacokinetic characteristics. Here, we review the co-crystal information of a selection of NPs, focusing on the SARs of NPs reflected by co-crystal information and the modification and simplification strategies of NPs, and discuss how to apply co-crystal information in the optimization of NP-based lead compound.
Subject(s)
Biological Products , Biological Products/pharmacology , Biological Products/chemistry , Drug Discovery/methods , Structure-Activity Relationship , CrystallographyABSTRACT
The relationship between urinary tract infection caused by urease-producing bacteria and lithiasis due to struvite stones is well established in the literature. However, there is limited knowledge on whether non-urease producing bacteria can also promote crystallization. In our study, we analyzed the association between urinary lithiasis, other than struvite by crystallography and non-ureolytic bacteria, in 153 patients who underwent surgery for urinary stone. The collected samples were sent for crystallographic analysis and culture. Additionally, preoperatory urine culture was collected for combined evaluation with the previous data. Percutaneous nephrolithotomy was the most commonly performed approach (45.8%). Struvite stones were more frequently identified in women (90.3%). Among stones with positive cultures, except struvite, 45.5% were composed of calcium oxalate monohydrate. The difference between urine culture and stone culture was different in 24.8% of the cases. Among stones with positive cultures that did not contain struvite, 86.4% showed non-urease bacteria in their cultures and 47.1% of struvite stones also did not have urease-producing bacteria in their cultures (p < 0.021). Our findings suggest that there is an association between non-ureolytic bacteria and stones that are not composed of struvite.
Subject(s)
Urinary Calculi , Urolithiasis , Humans , Female , Struvite , Crystallography , Urease , Urolithiasis/complications , Urinary Calculi/urine , BacteriaABSTRACT
Deep learning techniques can recognize complex patterns in noisy, multidimensional data. In recent years, researchers have started to explore the potential of deep learning in the field of structural biology, including protein crystallography. This field has some significant challenges, in particular producing high-quality and well ordered protein crystals. Additionally, collecting diffraction data with high completeness and quality, and determining and refining protein structures can be problematic. Protein crystallographic data are often high-dimensional, noisy and incomplete. Deep learning algorithms can extract relevant features from these data and learn to recognize patterns, which can improve the success rate of crystallization and the quality of crystal structures. This paper reviews progress in this field.
Subject(s)
Deep Learning , Crystallography , Algorithms , Compulsive Behavior , CrystallizationABSTRACT
Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump-probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.
Subject(s)
Proteins , Synchrotrons , Crystallography , Crystallography, X-Ray , X-Rays , Proteins/chemistry , LasersABSTRACT
Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.