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Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest. [ABSTRACT FROM AUTHOR]

Various X-ray techniques are employed to investigate specimens in diverse fields. Generally, scattering and absorption/emission processes occur due to the interaction of X-rays with matter. The output signals from these processes contain structural information and the electronic structure of specimens, respectively. The combination of complementary X-ray techniques improves the understanding of complex systems holistically. In this context, we introduce a multiplex imaging instrument that can collect small-/wide-angle X-ray diffraction and X-ray emission spectra simultaneously to investigate morphological information with nanoscale resolution, crystal arrangement at the atomic scale and the electronic structure of specimens. [ABSTRACT FROM AUTHOR]

P2X7 is an ATP-activated purinergic receptor implicated in pro-inflammatory responses. It is associated with the development of several diseases, including inflammatory and neurodegenerative conditions. Although several P2X7 receptor antagonists have recently been reported in the literature, none of them is approved for clinical use. However, the structure of the known antagonists can serve as a scaffold for discovering effective compounds in clinical therapy. This study aimed to propose an improved virtual screening methodology for the identification of novel potential P2X7 receptor antagonists from natural products through the combination of shape-based and docking approaches. First, a shape-based screening was performed based on the structure of JNJ-47965567, a P2X7 antagonist, using two natural product compound databases, MEGx (~5.8 × 103 compounds) and NATx (~32 × 103 compounds). Then, the compounds selected by the proposed shape-based model, with Shape–Tanimoto score values ranging between 0.624 and 0.799, were filtered for drug-like properties. Finally, the compounds that met the drug-like filter criteria were docked into the P2X7 allosteric binding site, using the docking programs GOLD and DockThor. The docking poses with the best score values were submitted to careful visual inspection of the P2X7 allosteric binding site. Based on our established visual inspection criteria, four compounds from the MEGx database and four from the NATx database were finally selected as potential P2X7 receptor antagonists. The selected compounds are structurally different from known P2X7 antagonists, have drug-like properties, and are predicted to interact with key P2X7 allosteric binding pocket residues, including F88, F92, F95, F103, M105, F108, Y295, Y298, and I310. Therefore, the combination of shape-based screening and docking approaches proposed in our study has proven useful in selecting potential novel P2X7 antagonist candidates from natural-product-derived compounds databases. This approach could also be useful for selecting potential inhibitors/antagonists of other receptors and/or biological targets. [ABSTRACT FROM AUTHOR]

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

Key messages: Narrowing down to a single putative target gene behind a leaf senescence mutant and constructing the regulation network by proteomic method. Leaf senescence mutant is an important resource for exploring molecular mechanism of aging. To dig for potential modulation networks during maize leaf aging process, we delimited the gene responsible for a premature leaf senescence mutant els5 to a 1.1 Mb interval in the B73 reference genome using a BC1F1 population with 40,000 plants, and analyzed the leaf proteomics of the mutant and its near-isogenic wild type line. A total of 1355 differentially accumulated proteins (DAP) were mainly enriched in regulation pathways such as "photosynthesis", "ribosome", and "porphyrin and chlorophyll metabolism" by the KEGG pathway analysis. The interaction networks constructed by incorporation of transcriptome data showed that ZmELS5 likely repaired several key factors in the photosynthesis system. The putative candidate proteins for els5 were proposed based on DAPs in the fined QTL mapping interval. These results provide fundamental basis for cloning and functional research of the els5 gene, and new insights into the molecular mechanism of leaf senescence in maize. [ABSTRACT FROM AUTHOR]

Proteins guide the flows of information, energy, and matter that make life possible by accelerating transport and chemical reactions, by allosterically modulating these reactions, and by forming dynamic supramolecular assemblies. In these roles, conformational change underlies functional transitions. Time-resolved X-ray diffraction methods characterize these transitions either by directly triggering sequences of functionally important motions or, more broadly, by capturing the motions of which proteins are capable. To date, most successful have been experiments in which conformational change is triggered in light-dependent proteins. In this review, I emphasize emerging techniques that probe the dynamic basis of function in proteins lacking natively light-dependent transitions and speculate about extensions and further possibilities. In addition, I review how the weaker and more distributed signals in these data push the limits of the capabilities of analytical methods. Taken together, these new methods are beginning to establish a powerful paradigm for the study of the physics of protein function. [ABSTRACT FROM AUTHOR]

Targeting RNA methyltransferases with small molecules as inhibitors or tool compounds is an emerging field of interest in epitranscriptomics and medicinal chemistry. For two challenging RNA methyltransferases that introduce the 5-methylcytosine (m5C) modification in different tRNAs, namely DNMT2 and NSUN6, an ultra-large commercially available chemical space was virtually screened by physicochemical property filtering, molecular docking, and clustering to identify new ligands for those enzymes. Novel chemotypes binding to DNMT2 and NSUN6 with affinities down to KD,app = 37 µM and KD,app = 12 µM, respectively, were identified using a microscale thermophoresis (MST) binding assay. These compounds represent the first molecules with a distinct structure from the cofactor SAM and have the potential to be developed into activity-based probes for these enzymes. Additionally, the challenges and strategies of chemical space docking screens with special emphasis on library focusing and diversification are discussed. [ABSTRACT FROM AUTHOR]

A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Timeresolved small- and wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump-probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and coflow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein. [ABSTRACT FROM AUTHOR]

There is an increasing demand for simple and efficient sample delivery technology to match the rapid development of serial crystallography and its wide application in analyzing the structural dynamics of biological macromolecules. Here, a microfluidic rotating-target device is presented, capable of three-degrees-of-freedom motion, including two rotational degrees of freedom and one translational degree of freedom, for sample delivery. Lysozyme crystals were used as a test model with this device to collect serial synchrotron crystallography data and the device was found to be convenient and useful. This device enables in situ diffraction from crystals in a microfluidic channel without the need for crystal harvesting. The circular motion ensures that the delivery speed can be adjusted over a wide range, showing its good compatibility with different light sources. Moreover, the three-degrees-of-freedom motion guarantees the full utilization of crystals. Hence, sample consumption is greatly reduced, and only 0.1 mg of protein is consumed in collecting a complete dataset. [ABSTRACT FROM AUTHOR]

The phototransduction cascade in vertebrate rod visual cells is initiated by the photoactivation of rhodopsin, which enables the activation of the visual G protein transducin. It is terminated by the phosphorylation of rhodopsin, followed by the binding of arrestin. Here we measured the solution X-ray scattering of nanodiscs containing rhodopsin in the presence of rod arrestin to directly observe the formation of the rhodopsin/arrestin complex. Although arrestin self-associates to form a tetramer at physiological concentrations, it was found that arrestin binds to phosphorylated and photoactivated rhodopsin at 1:1 stoichiometry. In contrast, no complex formation was observed for unphosphorylated rhodopsin upon photoactivation, even at physiological arrestin concentrations, suggesting that the constitutive activity of rod arrestin is sufficiently low. UV-visible spectroscopy demonstrated that the rate of the formation of the rhodopsin/arrestin complex well correlates with the concentration of arrestin monomer rather than the tetramer. These findings indicate that arrestin monomer, whose concentration is almost constant due to the equilibrium with the tetramer, binds to phosphorylated rhodopsin. The arrestin tetramer would act as a reservoir of monomer to compensate for the large changes in arrestin concentration in rod cells caused by intense light or adaptation. [ABSTRACT FROM AUTHOR]

Purple phototrophic bacteria use a 'photosystem' consisting of light harvesting complex 1 (LH1) surrounding the reaction centre (RC) that absorbs far-red-near-infrared light and converts it to chemical energy. Blastochloris species, which harvest light >1000 nm, use bacteriochlorophyll b rather than the more common bacteriochlorophyll a as their major photopigment, and assemble LH1 with an additional polypeptide subunit, LH1γ, encoded by multiple genes. To assign a role to γ, we deleted the four encoding genes in the model Blastochloris viridis. Interestingly, growth under halogen bulbs routinely used for cultivation yielded cells displaying an absorption maximum of 825 nm, similar to that of the RC only, but growth under white light yielded cells with an absorption maximum at 972 nm. HPLC analysis of pigment composition and sucrose gradient fractionation demonstrate that the white light-grown mutant assembles RC-LH1, albeit with an absorption maximum blue-shifted by 46 nm. Wavelengths between 900-1000 nm transmit poorly through the atmosphere due to absorption by water, so our results provide an evolutionary rationale for incorporation of γ; this polypeptide red-shifts absorption of RC-LH1 to a spectral range in which photons are of lower energy but are more abundant. Finally, we transformed the mutant with plasmids encoding natural LH1γ variants and demonstrate that the polypeptide found in the wild type complex red-shifts absorption back to 1018 nm, but incorporation of a distantly related variant results in only a moderate shift. This result suggests that tuning the absorption of RC-LH1 is possible and may permit photosynthesis past its current low-energy limit. [ABSTRACT FROM AUTHOR]

In the structure of photosynthetic reaction center (RC) of the purple bacterium Cereibacter sphaeroides the highly conserved amino acid residue Ile-M206 is located near the bacteriochlorophyll dimer P, which is the primary electron donor, and the monomeric bacteriochlorophyll BA, which is the nearest electron acceptor. Since Ile-M206 is close to the C2-acetyl group of bacteriochlorophyll PB, the hydroxyl group of Tyr-M210, and to the C9-keto group of bacteriochlorophyll BA, as well as to the water molecule near the latter group, this site can be used for introducing mutations in order to study mechanisms of primary photochemical processes in the RC. Previously it was shown that the Ile→Glu substitution at the M204 position (analog of M206 in the RC of C. sphaeroides) in the RC of the closely related purple non-sulfur bacterium Rhodobacter capsulatus significantly affected kinetics of the P+HA– state formation, whereas the M204 Ile→Gln substitution led to the loss of BChl BA molecule from the complex structure. In the present work, it is shown that the single I(M206)Q or double I(M206)Q + F(M208)A amino acid substitutions in the RC of C. sphaeroides do not change the pigment composition and do not markedly influence redox potential of the primary electron donor. However, substitution of Ile M206 by Gln affected positions and amplitudes of the absorption bands of bacteriochlorophylls, increased lifetime of the primary electron donor P* excited state from 3.1 ps to 22 ps, and decreased quantum yield of the P+QA– state formation to 60%. These data suggest significant changes in the pigment–protein interactions in the vicinity of the primary electron donor P and the nearest electron acceptor BA. A considerable decrease was also noticed in the resistance of the mutant RC to thermal denaturation, which was more pronounced in the RC with the double substitution I(M206)Q + F(M208)A. This was likely associated with the disruption of the dense packing of the protein near bacteriochlorophylls PB and BA. Possible reasons for different effects of identical mutations on the properties of two highly homologous RCs from closely related purple non-sulfur bacteria are discussed. [ABSTRACT FROM AUTHOR]

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity. The authors present an in-depth investigation of excited state dynamics and molecular mechanism of the voltage sensing in microbial rhodopsins. Using a combination of spectroscopic investigations and molecular dynamics simulations, the study proposes the voltage-modulated deprotonation of the chromophore as the key event in the voltage sensing. Thus, molecular constraints that may further improve the fluorescence quantum yield and the voltage sensitivity are presented. [ABSTRACT FROM AUTHOR]

The effects of the novel [CuL2]Br2 complex (L = bis{4H-1,3,5-triazino [2,1-b]benzothiazole-2-amine,4-(2-imidazole)}copper(II) bromide complex) on the photosystem II (PSII) activity of PSII membranes isolated from spinach were studied. The absence of photosynthetic oxygen evolution by PSII membranes without artificial electron acceptors, but in the presence of [CuL2]Br2, has shown that it is not able to act as a PSII electron acceptor. In the presence of artificial electron acceptors, [CuL2]Br2 inhibits photosynthetic oxygen evolution. [CuL2]Br2 also suppresses the photoinduced changes of the PSII chlorophyll fluorescence yield (FV) related to the photoreduction of the primary quinone electron acceptor, QA. The inhibition of both characteristic PSII reactions depends on [CuL2]Br2 concentration. At all studied concentrations of [CuL2]Br2, the decrease in the FM level occurs exclusively due to a decrease in Fv. [CuL2]Br2 causes neither changes in the F0 level nor the retardation of the photoinduced rise in FM, which characterizes the efficiency of the electron supply from the donor-side components to QA through the PSII reaction center (RC). Artificial electron donors (sodium ascorbate, DPC, Mn2+) do not cancel the inhibitory effect of [CuL2]Br2. The dependences of the inhibitory efficiency of the studied reactions of PSII on [CuL2]Br2 complex concentration practically coincide. The inhibition constant Ki is about 16 µM, and logKi is 4.8. As [CuL2]Br2 does not change the aromatic amino acids' intrinsic fluorescence of the PSII protein components, it can be proposed that [CuL2]Br2 has no significant effect on the native state of PSII proteins. The results obtained in the present study are compared to the literature data concerning the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes. [ABSTRACT FROM AUTHOR]

A method is proposed to reconstruct the 3D molecular structure from micrographs collected at just one sample tilt angle in the random conical tilt scheme in cryo‐electron microscopy. The method uses autocorrelation analysis on the micrographs to estimate features of the molecule which are invariant under certain nuisance parameters such as the positions of molecular projections in the micrographs. This enables the molecular structure to be reconstructed directly from micrographs, completely circumventing the need for particle picking. Reconstructions are demonstrated with simulated data and the effect of the missing‐cone region is investigated. These results show promise to reduce the size limit for single‐particle reconstruction in cryo‐electron microscopy. [ABSTRACT FROM AUTHOR]

Helium droplets represent a cold inert matrix, free of walls with outstanding properties to grow complexes and clusters at conditions that are perfect to simulate cold and dense regions of the interstellar medium. At sub‐Kelvin temperatures, barrierless reactions triggered by radicals or ions have been observed and studied by optical spectroscopy and mass spectrometry. The present review summarizes developments of experimental techniques and methods and recent results they enabled. [ABSTRACT FROM AUTHOR]

Sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), a member of the SERCA family, is expressed ubiquitously and transports Ca2+ into the sarco/endoplasmic reticulum using the energy provided by ATP binding and hydrolysis. The crystal structure of SERCA2b in its Ca2+‐ and ATP‐bound (E1∙2Ca2+‐ATP) state and cryo‐electron microscopy (cryo‐EM) structures of the protein in its E1∙2Ca2+‐ATP and Ca2+‐unbound phosphorylated (E2P) states have provided essential insights into how the overall conformation and ATPase activity of SERCA2b is regulated by the transmembrane helix 11 and the subsequent luminal extension loop, both of which are specific to this isoform. More recently, our cryo‐EM analysis has revealed that SERCA2b likely adopts open and closed conformations of the cytosolic domains in the Ca2+‐bound but ATP‐free (E1∙2Ca2+) state, and that the closed conformation represents a state immediately prior to ATP binding. This review article summarizes the unique mechanisms underlying the conformational and functional regulation of SERCA2b. [ABSTRACT FROM AUTHOR]

The newly constructed time-resolved atomic, molecular and optical science instrument (TMO) is configured to take full advantage of both linear accelerators at SLAC National Accelerator Laboratory, the copper accelerator operating at a repetition rate of 120 Hz providing high per-pulse energy as well as the superconducting accelerator operating at a repetition rate of about 1 MHz providing high average intensity. Both accelerators power a soft X-ray freeelectron laser with the new variable-gap undulator section. With this flexible light source, TMO supports many experimental techniques not previously available at LCLS and will have two X-ray beam focus spots in line. Thereby, TMO supports atomic, molecular and optical, strong-field and nonlinear science and will also host a designated new dynamic reaction microscope with a submicrometer X-ray focus spot. The flexible instrument design is optimized for studying ultrafast electronic and molecular phenomena and can take full advantage of the sub-femtosecond soft X-ray pulse generation program. [ABSTRACT FROM AUTHOR]

The appearance of oxygenic photosynthesis in cyanobacteria is a major event in evolution. It had an irreversible impact on the Earth, promoting the Great Oxygenation Event (GOE) ~2.4 billion years ago. Ancient cyanobacteria predating the GOE were Gloeobacter -type cells lacking thylakoids, which hosted photosystems in their cytoplasmic membrane. The driver of the GOE was proposed to be the transition from unicellular to filamentous cyanobacteria. However, the appearance of thylakoids expanded the photosynthetic surface to such an extent that it introduced a multiplier effect, which would be more coherent with an impact on the atmosphere. Primitive thylakoids self-organize as concentric parietal uninterrupted multilayers. There is no robust evidence for an origin of thylakoids via a vesicular-based scenario. This review reports studies supporting that hexagonal II-forming glucolipids and galactolipids at the periphery of the cytosolic membrane could be turned, within nanoseconds and without any external source of energy, into membrane multilayers. Comparison of lipid biosynthetic pathways shows that ancient cyanobacteria contained only one anionic lamellar-forming lipid, phosphatidylglycerol. The acquisition of sulfoquinovosyldiacylglycerol biosynthesis correlates with thylakoid emergence, possibly enabling sufficient provision of anionic lipids to trigger a hexagonal II-to-lamellar phase transition. With this non-vesicular lipid-phase transition, a framework is also available to re-examine the role of companion proteins in thylakoid biogenesis. [ABSTRACT FROM AUTHOR]

Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X‐ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light‐activated proteins are attractive targets for time‐resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light‐absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small‐scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time‐resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light‐activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time‐resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time‐resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods. [ABSTRACT FROM AUTHOR]

The interplay between life sciences and advancing technology drives a continuous cycle of chemical data growth; these data are most often stored in open or partially open databases. In parallel, many different types of algorithms are being developed to manipulate these chemical objects and associated bioactivity data. Virtual screening methods are among the most popular computational approaches in pharmaceutical research. Today, user-friendly web-based tools are available to help scientists perform virtual screening experiments. This article provides an overview of internet resources enabling and supporting chemical biology and early drug discovery with a main emphasis on web servers dedicated to virtual ligand screening and small-molecule docking. This survey first introduces some key concepts and then presents recent and easily accessible virtual screening and related target-fishing tools as well as briefly discusses case studies enabled by some of these web services. Notwithstanding further improvements, already available web-based tools not only contribute to the design of bioactive molecules and assist drug repositioning but also help to generate new ideas and explore different hypotheses in a timely fashion while contributing to teaching in the field of drug development. [ABSTRACT FROM AUTHOR]

Membrane proteins govern critical cellular processes and are central to human health and associated disease. Understanding of membrane protein function is obscured by the vast ranges of structural dynamics—both in the spatial and time regime—displayed in the protein and surrounding membrane. The membrane lipids have emerged as allosteric modulators of membrane protein function, which further adds to the complexity. In this review, we discuss several examples of membrane dependency. A particular focus is on how molecular dynamics (MD) simulation have aided to map membrane protein dynamics and how enhanced sampling methods can enable observing the otherwise inaccessible biological time scale. Also, time-resolved X-ray scattering in solution is highlighted as a powerful tool to track membrane protein dynamics, in particular when combined with MD simulation to identify transient intermediate states. Finally, we discuss future directions of how to further develop this promising approach to determine structural dynamics of both the protein and the surrounding lipids. [ABSTRACT FROM AUTHOR]

Ancient parchments record an immense part of our cultural heritage, having been used as the main written support material for centuries. Parchment easily undergoes biodeterioration, whose main signs are the so-called purple spots , which often lead to detachment of the superficial written layer. Up to recent years, several studies have been analyzing damaged parchments from different world's archives, trying to trace back the culprit of the purple spots. However, standard cultivation and early molecular techniques have been demonstrated to be unsuccessful, leading the parchment damage issue remaining unsolved for many years. Nowadays, some studies have explored the parchment biodeterioration dynamics by adopting a multidisciplinary approach combining standard microbiological methods with high-throughput molecular, chemical and physical techniques. This approach allowed an unprecedented level of knowledge on the complex dynamics of parchment biodeterioration. This mini review discusses the application of the combination of basic and high-throughput techniques to study historical parchments, highlighting the strengths and weaknesses of this approach. In particular, it focuses on how metagenomics has been paramount for the unequivocal identification of the microbial main actors of parchment biodeterioration and their dynamics, but also on how metagenomics may suffer the distortion inflict by the historical perspective on the analysis of ancient specimens. As a whole, this mini review aims to describe the scenario of information on parchment biodeterioration obtained so far by using the integration of metagenomic with recent chemical (Raman spectroscopy) and physical (Light Transmission Analysis) approaches, which might have key implications in the preservation of many ancient documents. [ABSTRACT FROM AUTHOR]

Elucidating the structural dynamics of small molecules and proteins in the liquid solution phase is essential to ensure a fundamental understanding of their reaction mechanisms. In this regard, time‐resolved X‐ray solution scattering (TRXSS), also known as time‐resolved X‐ray liquidography (TRXL), has been established as a powerful technique for obtaining the structural information of reaction intermediates and products in the liquid solution phase and is expected to be applied to a wider range of molecules in the future. A TRXL experiment is generally performed at the beamline of a synchrotron or an X‐ray free‐electron laser (XFEL) to provide intense and short X‐ray pulses. Considering the limited opportunities to use these facilities, it is necessary to verify the plausibility of a target experiment prior to the actual experiment. For this purpose, a program has been developed, referred to as S‐cube, which is short for a Solution Scattering Simulator. This code allows the routine estimation of the shape and signal‐to‐noise ratio (SNR) of TRXL data from known experimental parameters. Specifically, S‐cube calculates the difference scattering curve and the associated quantum noise on the basis of the molecular structure of the target reactant and product, the target solvent, the energy of the pump laser pulse and the specifications of the beamline to be used. Employing a simplified form for the pair‐distribution function required to calculate the solute–solvent cross term greatly increases the calculation speed as compared with a typical TRXL data analysis. Demonstrative applications of S‐cube are presented, including the estimation of the expected TRXL data and SNR level for the future LCLS‐II HE beamlines. [ABSTRACT FROM AUTHOR]

A broadband energy-chirped hard X-ray pulse has been demonstrated at the SwissFEL (free electron laser) with up to 4% bandwidth. We consider the characteristic parameters for analyzing the time dependence of stationary protein diffraction with energy-chirped pulses. Depending on crystal mosaic spread, convergence, and recordable resolution, individual reflections are expected to spend at least ≈ 50 attoseconds and up to ≈ 8 femtoseconds in reflecting condition. Using parameters for a chirped XFEL pulse obtained from simulations of 4% bandwidth conditions, ray-tracing simulations have been carried out to demonstrate the temporal streaking across individual reflections and resolution ranges for protein crystal diffraction. Simulations performed at a higher chirp (10%) emphasize the importance of chirp magnitude that would allow increased observation statistics for the temporal separation of individual reflections for merging and structure determination. Finally, we consider the fundamental limitation for obtaining time-dependent observations using chirped pulse diffraction. We consider the maximum theoretical time resolution achievable to be on the order of 50–200 as from the instantaneous bandwidth of the chirped SASE pulse. We then assess the ability to propagate ultrafast optical pulses for pump-probe cross-correlation under characteristic conditions of material dispersion; in this regard, the limiting factors for time resolution scale with crystal thickness. Crystals that are below a few microns in size will be necessary for subfemtosecond time resolution. [ABSTRACT FROM AUTHOR]

Genome-resolved environmental metagenomic sequencing has uncovered substantial previously unrecognized microbial diversity relevant for understanding the ecology and evolution of the biosphere, providing a more nuanced view of the distribution and ecological significance of traits including phototrophy across diverse niches. Recently, the capacity for bacteriochlorophyll-based anoxygenic photosynthesis has been proposed in the uncultured bacterial WPS-2 phylum (recently proposed as Candidatus Eremiobacterota) that are in close association with boreal moss. Here, we use phylogenomic analysis to investigate the diversity and evolution of phototrophic WPS-2. We demonstrate that phototrophic WPS-2 show significant genetic and metabolic divergence from other phototrophic and non-phototrophic lineages. The genomes of these organisms encode a new family of anoxygenic Type II photochemical reaction centers and other phototrophy-related proteins that are both phylogenetically and structurally distinct from those found in previously described phototrophs. We propose the name Candidatus Baltobacterales for the order-level aerobic WPS-2 clade which contains phototrophic lineages, from the Greek for "bog" or "swamp," in reference to the typical habitat of phototrophic members of this clade. [ABSTRACT FROM AUTHOR]

Directional transport of protons across an energy transducing membrane—proton pumping—is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive. [ABSTRACT FROM AUTHOR]

A review that summarizes the most recent technological developments in the field of ultrafast structural dynamics with focus on the use of ultrashort X-ray and electron pulses follows. Atomistic views of chemical processes and phase transformations have long been the exclusive domain of computer simulators. The advent of femtosecond (fs) hard X-ray and fs-electron diffraction techniques made it possible to bring such a level of scrutiny to the experimental area. The following review article provides a summary of the main ultrafast techniques that enabled the generation of atomically resolved movies utilizing ultrashort X-ray and electron pulses. Recent advances are discussed with emphasis on synchrotron-based methods, tabletop fs-X-ray plasma sources, ultrabright fs-electron diffractometers, and timing techniques developed to further improve the temporal resolution and fully exploit the use of intense and ultrashort X-ray free electron laser (XFEL) pulses. [ABSTRACT FROM AUTHOR]

Blue‐absorbing proteorhodopsin (BPR) from marine bacteria is a retinal‐bound, light‐activated, outwards proton transporter containing seven α‐helical transmembrane segments (TMS). It is synthesized as a precursor species (pre‐BPR) with a predicted N‐terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre‐BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre‐BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild‐type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial "pro" domain rather than a traditional secretory peptide. Blue‐absorbing proteorhodopsins (BPR) are light‐activated proton transporters that reside in the inner membrane of marine bacteria but are synthesized with a cleavable signal sequence typical of secretory proteins. Here we show that although it improves yields, the signal sequence is not required for BPR to traffic to the plasma membrane, and that a variant with an uncleavable signal sequence is fully functional and accumulates at higher levels than the wild type protein in the membranes of E. coli. [ABSTRACT FROM AUTHOR]

The fundamental aim of structural analyses in biophysics is to reveal a mutual relation between a molecule's dynamic structure and its physiological function. Small-angle X-ray scattering (SAXS) is an experimental technique for structural characterization of macromolecules in solution and enables time-resolved analysis of conformational changes under physiological conditions. As such experiments measure spatially averaged low-resolution scattering intensities only, the sparse information obtained is not sufficient to uniquely reconstruct a three-dimensional atomistic model. Here, we integrate the information from SAXS into molecular dynamics simulations using computationally efficient native structure-based models. Dynamically fitting an initial structure towards a scattering intensity, such simulations produce atomistic models in agreement with the target data. In this way, SAXS data can be rapidly interpreted while retaining physico-chemical knowledge and sampling power of the underlying force field. We demonstrate our method's performance using the example of three protein systems. Simulations are faster than full molecular dynamics approaches by more than two orders of magnitude and consistently achieve comparable accuracy. Computational demands are reduced sufficiently to run the simulations on commodity desktop computers instead of high-performance computing systems. These results underline that scattering-guided structure-based simulations provide a suitable framework for rapid early-stage refinement of structures towards SAXS data with particular focus on minimal computational resources and time. [ABSTRACT FROM AUTHOR]

Light‐harvesting‐1 (LH1)‐reaction center (RC) super‐complex is a membrane protein–pigment complex existing in purple photosynthetic bacteria, where LH1 absorbs light energy and transfers them rapidly and efficiently to RC to initiate the charge separation and electron transfer reactions. The structure of LH1‐RC has been reported at relatively low resolutions from several different species of bacteria previously, but was solved at an atomic resolution recently from a thermophilic photosynthetic bacterium Thermochromatium tepidum. This high‐resolution structure revealed the detailed organization of the super‐complex including a number of unique features that are important for its functioning, such as a more intact RC structure, transporting routes for quinones to replace the bound QB as well as for the in‐and‐out of the closed LH1 ring, detailed coordinating environment of the Ca2+ ions in LH1 important for the remarkable red shift of the absorption spectrum, as well as for the enhanced thermostability. These results thus greatly advance our understanding on the mechanisms of energy transfer, quinone exchange, the red shift in the LH1‐Qy transition and the enhanced thermal stability, in this super‐complex. Crystal structure of the light‐harvesting 1‐reaction center super‐complex from a photosynthetic purple bacterium at an atomic resolution reveals a number of novel features regarding the overall organization and detailed arrangement of pigments and cofactors, quinone exchange and proton transfer channels, and the coordination pattern of Ca2+ ions important for red shift and thermostability of the super‐complex. [ABSTRACT FROM AUTHOR]

Recent observation of quantum vortices in superfluid 4He droplets measuring a few hundreds of nanometers in diameter involved decoration of vortex cores by clusters containing large numbers of Xe atoms, which served as X-ray contrast agents. Here, we report on the study of the kinematics of the combined vortex-cluster system in a cylinder and in a sphere. Equilibrium states, characterized by total angular momentum, L, were found by minimizing the total energy, E, which sums from the kinetic energy of the liquid due to the vortex and due to orbiting Xe clusters, as well as solvation energy of the cluster in the droplet. Calculations show that, at small mass of the cluster, the equilibrium displacement of the system from the rotation axis is close to that for the bare vortex. However, upon decrease in L beyond certain critical value, which is larger for heavier clusters, the displacement bifurcates toward the surface region, where the motion of the system is governed by the clusters. In addition, at even smaller L, bare orbiting clusters become energetically favorable, opening the possibility for the vortex to detach from the cluster and to annihilate at the droplet’s surface. [ABSTRACT FROM AUTHOR]

Proteorhodopsin is a light-driven proton pumping membrane protein related to bacteriorhodopsin. It contains an all- trans retinal A1 chromophore covalently bound to a lysine residue via a protonated Schiff base. In this study, we exploited density functional theory (DFT) calculations to investigate the retinal binding pocket in the dark state and after mimicking photoisomerization. The model of the binding pocket is constructed incrementally by adding the residues near the retinal that are necessary to ensure a stable protonated Schiff base. The presence of a few water molecules near the Schiff base turns out to be an essential feature of the model. The absorption properties are then studied using time-dependent DFT (TDDFT) and compared to experimental data to further validate the structural model and to assess the accuracy of the computational setting. It is shown that TDDFT is able to reproduce the main absorption peak accurately and to quantitatively determine the spectral shift induced by substituting the native all- trans retinal A1 chromophore with different retinal analogues. Moreover, ab initio molecular dynamics simulations are performed to investigate the vibrational spectra of our models before and after isomerization. Specific differences in the vibrational spectra are identified that provide further insight into experimental FTIR difference spectra. [ABSTRACT FROM AUTHOR]

Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases. [ABSTRACT FROM AUTHOR]

The broad range of applications of synchrotron and neutron scattering in the investigation of soft condensed matter is reviewed. Appropriate combinations of these techniques allow probing the structure and dynamics of these complex systems from sub-nm to micron size scales and picoseconds to seconds and longer time ranges. Applications include a myriad of systems such as polymers, colloids, surfactants, phospholipids, biological macromolecules and functional materials both in bulk and at interfaces. Most studies are performed in situ under the real thermodynamic state of the given system and large ensemble averaged information is readily obtained. The new generations of synchrotron and neutron sources open possibilities for investigating more complex soft matter systems in hitherto unexplored dynamical states. [ABSTRACT FROM PUBLISHER]

Toward the end of his career, Zewail developed strong interest in fast electron spectroscopy and imaging, a field to which he made important contributions toward his aim of making molecular movies free of radiation damage. We therefore compare here the atomistic mechanisms leading to destruction of protein samples in diffract-and-destroy experiments for the cases of high-energy electron beam irradiation and X-ray laser pulses. The damage processes and their time-scales are compared and relevant elastic, inelastic, and photoelectron cross sections are given. Inelastic mean-free paths for ejected electrons at very low energies in insulators are compared with the bioparticle size. The dose rate and structural damage rate for electrons are found to be much lower, allowing longer pulses, reduced beam current, and Coulomb interactions for the formation of smaller probes. High-angle electron scattering from the nucleus, which has no parallel in the X-ray case, tracks the slowly moving nuclei during the explosion, just as the gain of the XFEL (X-ray free-electron laser) has no parallel in the electron case. Despite reduced damage and much larger elastic scattering cross sections in the electron case, leading to not dissimilar elastic scattering rates (when account is taken of the greatly increased incident XFEL fluence), progress for single-particle electron diffraction is seen to depend on the effort to reduce emittance growth due to Coulomb interactions, and so allow formation of intense sub-micron beams no larger than a virus. [ABSTRACT FROM AUTHOR]

In heme proteins, the efficient transport of ligands such as NO or O2 to the binding site is achieved via ligand migration networks. A quantitative assessment of ligand diffusion in these networks is thus essential for a better understanding of the function of these proteins. For this, Xe migration in truncated hemoglobin N (trHbN) of Mycobacterium Tuberculosis was studied using molecular dynamics simulations. Transitions between pockets of the migration network and intra-pocket relaxation occur on similar time scales (10 ps and 20 ps), consistent with low free energy barriers (1-2 kcal/mol). Depending on the pocket from where Xe enters a particular transition, the conformation of the side chains lining the transition region differs which highlights the coupling between ligand and protein degrees of freedom. Furthermore, comparison of transition probabilities shows that Xe migration in trHbN is a non-Markovian process. Memory effects arise due to protein rearrangements and coupled dynamics as Xe moves through it. [ABSTRACT FROM AUTHOR]

Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background. [ABSTRACT FROM AUTHOR]

Despite significant progress made over more than 15 years of research, structural biologists are still grappling with the issue of radiation damage suffered by macromolecular crystals which is induced by the resultant radiation chemistry occurring during X-ray diffraction experiments. Further insights into these effects and the possible mitigation strategies for use in both diffraction and SAXS experiments are given in eight papers in this volume. In particular, damage during experimental phasing is addressed, scavengers for SAXS experiments are investigated, microcrystals are imaged, data collection strategies are optimized, specific damage to tyrosine residues is reexamined, and room temperature conformational heterogeneity as a function of dose is explored. The brief summary below puts these papers into perspective relative to other ongoing radiation damage research on macromolecules. [ABSTRACT FROM AUTHOR]

The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free-electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future. [ABSTRACT FROM AUTHOR]

"Protein quake" denotes the dissipation of excess energy across a protein, in response to a local perturbation such as the breaking of a chemical bond or the absorption of a photon. Femtosecond timeresolved small- and wide-angle X-ray scattering (TR-SWAXS) is capable of tracking such ultrafast protein dynamics. However, because the structural interpretation of the experiments is complicated, a molecular picture of protein quakes has remained elusive. In addition, new questions arose from recent TR-SWAXS data that were interpreted as underdamped oscillations of an entire protein, thus challenging the long-standing concept of overdamped global protein dynamics. Based on molecular-dynamics simulations, we present a detailed molecular movie of the protein quake after carbon monoxide (CO) photodissociation in myoglobin. The simulations suggest that the protein quake is characterized by a single pressure peak that propagates anisotropically within 500 fs across the protein and further into the solvent. By computing TR-SWAXS patterns from the simulations, we could interpret features in the reciprocalspace SWAXS signals as specific real-space dynamics, such as CO displacement and pressure wave propagation. Remarkably, we found that the small-angle data primarily detect modulations of the solvent density but not oscillations of the bare protein, thereby reconciling recent TR-SWAXS experiments with the notion of overdamped global protein dynamics. [ABSTRACT FROM AUTHOR]

The hard X-ray laser SwissFEL at the Paul Scherrer Institute is currently being commissioned and will soon become available for users. In the current article the laser facility is presented, an integral part of the user facility, as most time-resolved experiments will require a versatile optical laser infrastructure and precise information about the relative delay between the X-ray and optical pulse. The important key parameters are a high availability and long-term stability while providing advanced laser performance in the wavelength range from ultraviolet to terahertz. The concept of integrating a Ti:sapphire laser amplifier system with subsequent frequency conversion stages and drift compensation into the SwissFEL facility environment for successful 24 h/7 d user operation is described. [ABSTRACT FROM AUTHOR]

X-ray crystallography promises direct insights into electron-density changes that lead to and arise from structural changes such as electron and proton transfer and the formation, rupture and isomerization of chemical bonds. The ultrashort pulses of hard X-rays produced by free-electron lasers present an exciting opportunity for capturing ultrafast structural events in biological macro-molecules within femtoseconds after photoexcitation. However, shot-to-shot fluctuations, which are inherent to the very process of self-amplified spontaneous emission (SASE) that generates the ultrashort X-ray pulses, are a major source of noise that may conceal signals from structural changes. Here, a new approach is proposed to angularly split a single SASE pulse and to produce a temporal delay of picoseconds between the split pulses. These split pulses will allow the probing of two distinct states before and after photoexcitation triggered by a laser pulse between the split X-ray pulses. The split pulses originate from a single SASE pulse and share many common properties; thus, noise arising from shot-to-shot fluctuations is self-canceling. The unambiguous interpretation of ultrafast structural changes would require diffraction data at atomic resolution, as these changes may or may not involve any atomic displacement. This approach, in combination with the strategy of serial crystallography, offers a solution to study ultrafast dynamics of light-initiated biochemical reactions or biological processes at atomic resolution. [ABSTRACT FROM AUTHOR]

In experimental research referencing two or more measurements to one another is a powerful tool to reduce the effect of systematic errors between different sets of measurements. The interesting quantity is usually derived from two measurements on the same sample under different conditions. While an elaborate experimental design is essential for improving the estimate, the data analysis should also maximally exploit the covariance between the measurements. In X-ray crystallography the difference between structure-factor amplitudes carries important information to solve experimental phasing problems or to determine time-dependent structural changes in pump-probe experiments. Here a multivariate Bayesian method was used to analyse intensity measurement pairs to determine their underlying structure-factor amplitudes and their differences. The posterior distribution of the model parameter was approximated with a Markov chain Monte Carlo algorithm. The described merging method is shown to be especially advantageous when systematic and random errors result in recording negative intensity measurements. [ABSTRACT FROM AUTHOR]

Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. [ABSTRACT FROM AUTHOR]

A new class of femtosecond, intense, short – wavelength lasers – the free-electron laser – has opened up new opportunities to investigate the structure and dynamics in many scientific areas. These new lasers, whose performance keeps increasing, enable the understanding of physical and chemical changes at an atomic spatial scale and on the time scale of atomic motion which is essential for a broad range of scientific fields. We describe here the interaction of fullerenes in the multiphoton regime with the Linac Coherent Light Source (LCLS) X-ray free-electron laser at SLAC National Laboratory. In particular, we report on new data regarding the ionization of Ho3N@C80molecules and compare the results with our prior C60investigation of radiation damage induced by the LCLS pulses. We also discuss briefly the potential impact of newly available instrumentation to physical and chemical sciences when they are coupled with FELs as well as theoretical calculations and modeling. [ABSTRACT FROM AUTHOR]