Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

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.

Advances and applications of microcrystal electron diffraction (MicroED).

Microcrystal electron diffraction, commonly referred to as MicroED, has become a powerful tool for high-resolution structure determination. The method makes use of cryogenic transmission electron microscopes to collect electron diffraction data from crystals that are several orders of magnitude smaller than those used by other conventional diffraction techniques. MicroED has been used on a variety of samples including soluble proteins, membrane proteins, small organic molecules, and materials. Here we will review the MicroED method and highlight recent advancements to the methodology, as well as describe applications of MicroED within the fields of structural biology and chemical crystallography.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Review of serial femtosecond crystallography including the COVID-19 pandemic impact and future outlook.

Serial femtosecond crystallography (SFX) revolutionized macromolecular crystallography over the past decade by enabling the collection of X-ray diffraction data from nano- or micrometer sized crystals while outrunning structure-altering radiation damage effects at room temperature. The serial manner of data collection from millions of individual crystals coupled with the femtosecond duration of the ultrabright X-ray pulses enables time-resolved studies of macromolecules under near-physiological conditions to unprecedented temporal resolution. In 2020 the rapid spread of the coronavirus SARS-CoV-2 resulted in a global pandemic of coronavirus disease-2019. This led to a shift in how serial femtosecond experiments were performed, along with rapid funding and free electron laser beamtime availability dedicated to SARS-CoV-2-related studies. This review outlines the current state of SFX research, the milestones that were achieved, the impact of the global pandemic on this field as well as an outlook into exciting future directions.

Applications and limitations of electron 3D crystallography.

Three-dimensional electron diffraction (3D ED) is a measurement and analysis technique in transmission electron microscopy that is used for determining atomic structures from small crystals. Diverse targets such as proteins, polypeptides, and organic compounds, whose crystals exist in aqueous solutions and organic solvents, or as dried powders, can be studied with 3D ED. We have been involved in the development of this technique, which can now rapidly process a large number of data collected through AI control, enabling efficient structure determination. Here, we introduce this method and describe our recent results. These include the structures and pathogenic mechanisms of wild-type and mutant polypeptides associated with the debilitating disease amyotrophic lateral sclerosis (ALS), the double helical structure of nanographene promoting nanofiber formation, and the structural properties of an organic semiconductor containing disordered regions. We also discuss the limitations and prospects of 3D ED compared to microcrystallography with X-ray free electron lasers.

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography.

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

Sample Preparation for Time-Resolved Serial Crystallography: Practical Considerations.

Time-resolved serial crystallography is an emerging method to elucidate the structure-function relationship of biomolecular systems at up to atomic resolution. However, to make this demanding method a success, a number of experimental requirements have to be met. In this chapter, we summarize general guidelines and protocols towards performing time-resolved crystallography experiments, with a particular emphasis on sample requirements and preparation but also a brief excursion into reaction initiation.

Preparation of RNA Microcrystals for Serial Femtosecond Crystallography Experiments.

Serial femtosecond crystallography (SFX) experiments using an X-ray free electron laser (XFEL) is a burgeoning method for time-resolved structural studies of biomacromolecules. As with any crystallography experiment, the most important component is quality sample preparation. Whereas dozens of SFX experiments, including batch crystallization methods, have been reported for proteins, very few have been reported for RNA. This chapter outlines standard procedures for preparing RNA microcrystalline samples suitable for SFX studies.

Mix-and-Inject Serial Femtosecond Crystallography to Capture RNA Riboswitch Intermediates.

Time-resolved structure determination of macromolecular conformations and ligand-bound intermediates is extremely challenging, particularly for RNA. With rapid technological advances in both microfluidic liquid injection and X-ray free electron lasers (XFEL), a new frontier has emerged in time-resolved crystallography whereby crystals can be mixed with ligand and then probed with X-rays (mix-and-inject) in real time and at room temperature. This chapter outlines the basic setup and procedures for mix-and-inject experiments for recording time-resolved crystallographic data of riboswitch RNA reaction states using serial femtosecond crystallography (SFX) and an XFEL.

Triple helix and rod structures of the antiseptic drug bibrocathol revealed by electron crystallography.

Bibrocathol is an active pharmaceutical ingredient that has been used to treat eyelid diseases for over a century, yet its structure has remained unknown. 3D electron diffraction on crystals from a commercial ointment revealed two structures. These results highlight the technique's potential in structure elucidation from microcrystalline mixtures.

Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies.

Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, which hinders structure determination by conventional methods. To automatically extract structural information pertaining to these states, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of methods hitherto not available; (iii) calculates extrapolated structure factors using the various proposed formalisms while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures in real and reciprocal-space. The use of Xtrapol8 could accelerate data processing in kinetic and time-resolved crystallographic studies, and as well foster the identification of drug-targetable states in ligand-based crystallography.

Pre- and Post-publication Verification for Reproducible Data Mining in Macromolecular Crystallography.

Like an article narrative is deemed by an editor and referees to be worthy of being a version of record on acceptance as a publication, so must the underpinning data also be scrutinized before passing it as a version of record. Indeed without the underpinning data, a study and its conclusions cannot be reproduced at any stage of evaluation, pre- or post-publication. Likewise, an independent study without its own underpinning data also cannot be reproduced let alone be considered a replicate of the first study. The PDB is a modern marvel of achievement providing an organized open access to depositor and user of the data held there opening numerous applications. Methods for modeling protein structures and for determination of structures are still improving their precision, and artifacts of the method exist. So their accuracy is realized if they are reproduced by other methods. It is on such foundations that reproducible data mining is based. Data rates are expanding considerably be they at synchrotrons, the X-ray free electron lasers (XFELs), electron cryomicroscopes (cryoEM), or at the neutron facilities. The work of a person as a referee or user with a narrative and its underpinning data may well be complemented in future by artificial intelligence with machine learning, the former for specific refereeing and the latter for the more general validation, both ideally before publication. Examples are described involving rhenium theranostics, the anti-cancer platins and the SARS-CoV-2 main protease.

An Outline of the Latest Crystallographic Studies on Inhibitor-Enzyme Complexes for the Design and Development of New Therapeutics against Tuberculosis.

The elucidation of the structure of enzymes and their complexes with ligands continues to provide invaluable insights for the development of drugs against many diseases, including bacterial infections. After nearly three decades since the World Health Organization's (WHO) declaration of tuberculosis (TB) as a global health emergency, Mycobacterium tuberculosis (Mtb) continues to claim millions of lives, remaining among the leading causes of death worldwide. In the last years, several efforts have been devoted to shortening and improving treatment outcomes, and to overcoming the increasing resistance phenomenon. The structural elucidation of enzyme-ligand complexes is fundamental to identify hot-spots, define possible interaction sites, and elaborate strategies to develop optimized molecules with high affinity. This review offers a critical and comprehensive overview of the most recent structural information on traditional and emerging mycobacterial enzymatic targets. A selection of more than twenty enzymes is here discussed, with a special emphasis on the analysis of their binding sites, the definition of the structure-activity relationships (SARs) of their inhibitors, and the study of their main intermolecular interactions. This work corroborates the potential of structural studies, substantiating their relevance in future anti-mycobacterial drug discovery and development efforts.

Heterogeneous Composition of Oxygen-Evolving Complexes in Crystal Structures of Dark-Adapted Photosystem II.

Photosystem II (PSII) is a homodimeric protein complex that catalyzes water oxidation at the oxygen-evolving complex (OEC), a heterocubanoid calcium-tetramanganese cluster. Here, we analyze the omit electron density peaks of the OEC's metal ions in five X-ray free-electron laser PSII structures at resolutions between 2.15 and 1.95 Å. The omit peaks can be described by the total number of electrons and approximated by the variance of electron density distribution when the distributions are spherically symmetric. We show that the number of electrons of metal centers is different in the two OECs of PSII dimers, implying that the oxidation states and/or occupancies of individual metal ions are different in the two monomers. In either case, we find that the two OECs of dark-adapted PSII dimers in crystals are not fully synchronized in the S1 state. Differences in redox states of the OEC in PSII only partially account for the observation that the electron densities integrate to a smaller number of electrons than expected. Differences between the determined and expected relative electron numbers are much larger than the estimated errors, indicating heterogeneity in the OEC composition.

Imaging of bee honey sugar crystals by second-harmonic generation microscopy.

Bee honey is an exceptionally nutritious food with unique chemical and mineral contents. This report introduces the use of the second-harmonic generation (SHG) microscopy for imaging honey sugar crystals' morphology as an alternative for its authentication process. The crystals and their boundaries are clearly observed with SHG compared with bright-field microscopy, where the liquid honey avoids the visualization of a sharp image. Four different honey samples of Mexico's various floral origins and geographical regions are analyzed in our study. These samples are representative of the diversity and valuable quality of bee honey production. The SHG image information is complemented with Raman spectroscopy (RS) analysis, since this optical technique is widely used to validate the bee's honey composition stated by its floral origin. We relate the SHG imaging of honey crystals with the well-defined fructose and glucose peaks measured by RS. Size measurement is introduced using the crystal´s length ratio to differentiate its floral origin. From our observations, we can state that SHG is a promising and suitable technique to provide a sort of optical fingerprint based on the floral origin of bee honey.

Enhancing protein perdeuteration by experimental evolution of Escherichia coli K-12 for rapid growth in deuterium-based media.

Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.

Importance of Nuclear Quantum Effects for NMR Crystallography.

The resolving power of solid-state nuclear magnetic resonance (NMR) crystallography depends heavily on the accuracy of computational predictions of NMR chemical shieldings of candidate structures, which are usually taken to be local minima in the potential energy. To test the limits of this approximation, we systematically study the importance of finite-temperature and quantum nuclear fluctuations for 1H, 13C, and 15N shieldings in polymorphs of three paradigmatic molecular crystals: benzene, glycine, and succinic acid. The effect of quantum fluctuations is comparable to the typical errors of shielding predictions for static nuclei with respect to experiments, and their inclusion improves the agreement with measurements, translating to more reliable assignment of the NMR spectra to the correct candidate structure. The use of integrated machine-learning models, trained on first-principles energies and shieldings, renders rigorous sampling of nuclear fluctuations affordable, setting a new standard for the calculations underlying NMR structure determinations.

The SARS-CoV-2 Programmed -1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography.

The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

Molecular recognition of a single-chain Fv antibody specific for GA-pyridine, an advanced glycation end-product (AGE), elucidated using biophysical techniques and synthetic antigen analogues.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.