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The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.

Ever since the first structure of an enzyme, lysozyme, was solved, scientists have been eager to explore how these molecules perform their catalytic function. There has been an overwhelmingly large body of publications that report the X-ray structures of enzymes determined after substrate and ligand binding. None of them truly show the structures of an enzyme working freely through a sequence of events that range from the formation of the enzyme–substrate complex to the dissociation of the product. The technical difficulties were too severe. By 1969, Sluyterman and de Graaf had pointed out that there might be a way to start a reaction in an enzyme crystal by diffusion and following its catalytic cycle in its entirety with crystallographic methods. The crystal only has to be thin enough so that the diffusion is not rate limiting. Of course, the key questions are as follows: How thin should the crystal be? Will the existing X-ray sources be able to collect data from a thin enough crystal fast enough? This review shines light on these questions.

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

Inspired by recent progress in time-resolved x-ray crystallography and the adoption of time-resolution by cryo-electronmicroscopy, this article enumerates several approaches developed to become bigger/smaller, faster, and better to gain new insight into the molecular mechanisms of life. This is illustrated by examples where chemical and physical stimuli spawn biological responses on various length and time-scales, from fractions of Ångströms to micro-meters and from femtoseconds to hours.

Time-resolved macromolecular crystallography unifies structure determination with chemical kinetics, since the structures of transient states and chemical and kinetic mechanisms can be determined simultaneously from the same data. To start a reaction in an enzyme, typically, an initially inactive substrate present in the crystal is activated. This has particular disadvantages that are circumvented when active substrate is directly provided by diffusion. However, then it is prohibitive to use macroscopic crystals because diffusion times become too long. With small micro- and nanocrystals diffusion times are adequately short for most enzymes and the reaction can be swiftly initiated. We demonstrate here that a time-resolved crystallographic experiment becomes feasible by mixing substrate with enzyme nanocrystals which are subsequently injected into the X-ray beam of a pulsed X-ray source.

Monitoring of canopy density with related metrics such as leaf area index (LAI) makes a significant contribution to understanding and predicting processes in the soil–plant–atmosphere system and to indicating crop health and potential yield for farm management. Remote sensing methods using optical sensors that rely on spectral reflectance to calculate LAI have become more mainstream due to easy entry and availability. Methods with vegetation indices (VI) based on multispectral reflectance data essentially measure the green area index (GAI) or response to chlorophyll content of the canopy surface and not the entire aboveground biomass that may be present from non-green elements that are key to fully assessing the carbon budget. Methods with light detection and ranging (LiDAR) have started to emerge using gap fraction (GF) to estimate the plant area index (PAI) based on canopy density. These LiDAR methods have the main advantage of being sensitive to both green and non-green plant elements. They have primarily been applied to forest cover with manned airborne LiDAR systems (ALS) and have yet to be used extensively with crops such as winter wheat using LiDAR on unmanned aircraft systems (UAS). This study contributes to a better understanding of the potential of LiDAR as a tool to estimate canopy structure in precision farming. The LiDAR method proved to have a high to moderate correlation in spatial variation to the multispectral method. The LiDAR-derived PAI values closely resemble the SunScan Ceptometer GAI ground measurements taken early in the growing season before major stages of senescence. Later in the growing season, when the canopy density was at its highest, a possible overestimation may have occurred. This was most likely due to the chosen flight parameters not providing the best depictions of canopy density with consideration of the LiDAR’s perspective, as the ground-based destructive measurements provided lower values of PAI. Additionally, a distinction between total LiDAR-derived PAI, multispectral-derived GAI, and brown area index (BAI) is made to show how the active and passive optical sensor methods used in this study can complement each other throughout the growing season.

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology. The characterization of biomacromolecule structural vibrations has been impeded by a broad continuous vibrational density of states obscuring molecule specific vibrations. A terahertz microscopy system using polarization control produces signatures to dynamically fingerprint proteins and a RNA G-quadruplex.

This is the second, and last paper in which we address the behavior of oriented first passage percolation on the hypercube in the limit of large dimensions. We prove here that the extremal process converges to a Cox process with exponential intensity. This entails, in particular, that the first passage time converges weakly to a random shift of the Gumbel distribution. The random shift, which has an explicit, universal distribution related to modified Bessel functions of the second kind, is the sole manifestation of correlations ensuing from the geometry of Euclidean space in infinite dimensions. The proof combines the multiscale refinement of the second moment method with a conditional version of the Chen–Stein bounds, and a contraction principle.

Trans -to- cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p -coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (I T ) resembles a highly strained transition state in which the torsion angle is located halfway between the trans - and cis- isomers. The reaction trajectory of I T bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway. Time-resolved X-ray crystallography on photoactive yellow protein shows the existence of a short-lived, highly distorted intermediate whose reaction trajectory bifurcates along ‘bicycle-pedal’ and ‘hula-twist’ pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue, which switches off the bicycle-pedal pathway.

A 9-nm-resolution structure of PR772 virus and a movie of its continuous conformational changes are determined from single-particle X-ray scattering data. Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure from a portal vertex and release of the genome. These results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes.