Time-resolved crystallography and protein design: signalling photoreceptors and optogenetics

Time-resolved X-ray crystallography and solution scattering have been successfully conducted on proteins on time-scales down to around 100 ps, set by the duration of the hard X-ray pulses emitted by synchrotron sources. The advent of hard X-ray free-electron lasers (FELs), which emit extremely intense, very brief, coherent X-ray pulses, opens the exciting possibility of time-resolved experiments with femtosecond time resolution on macromolecular structure, in both single crystals and solution. The X-ray pulses emitted by an FEL differ greatly in many properties from those emitted by a synchrotron, in ways that at first glance make time-resolved measurements of X-ray scattering with the required accuracy extremely challenging. This opens up several questions which I consider in this brief overview. Are there likely to be chemically and biologically interesting structural changes to be revealed on the femtosecond time-scale? How shall time-resolved experiments best be designed and conducted to exploit the properties of FELs and overcome challenges that they pose? To date, fast time-resolved reactions have been initiated by a brief laser pulse, which obviously requires that the system under study be light-sensitive. Although this is true for proteins of the visual system and for signalling photoreceptors, it is not naturally the case for most interesting biological systems. To generate more biological targets for time-resolved study, can this limitation be overcome by optogenetic, chemical or other means?     

Structural biology at the European X-ray free-electron laser facility

The European X-ray free-electron laser (XFEL) facility, under construction in the Hamburg region, will provide high-peak brilliance (greater than 10³³ photons s⁻¹ mm⁻² mrad⁻² per 0.1% BW), ultrashort pulses (approx. 10 fs) of X-rays, with a high repetition rate (up to 27 000 pulses s⁻¹) from 2016 onwards. The main features of this exceptional X-ray source, and the instrumentation developments necessary to exploit them fully, for application to a variety of scientific disciplines, are briefly summarized. In the case of structural biology, that has a central role in the scientific case of this new facility, the instruments and ancillary laboratories that are being planned and built within the baseline programme of the European XFEL and by consortia of users are also discussed. It is expected that the unique features of the source and the advanced features of the instrumentation will allow operation modes with more efficient use of sample materials, faster acquisition times, and conditions better approaching feasibility of single molecule imaging.     

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

BackgroundThe invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach.Scope of reviewRecent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described.Major conclusionsXFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data.General significanceXFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing ‘live’ protein molecules.     

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.     

Methods development for diffraction and spectroscopy studies of metalloenzymes at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) open up new possibilities for X-ray crystallographic and spectroscopic studies of radiation-sensitive biological samples under close to physiological conditions. To facilitate these new X-ray sources, tailored experimental methods and data-processing protocols have to be developed. The highly radiation-sensitive photosystem II (PSII) protein complex is a prime target for XFEL experiments aiming to study the mechanism of light-induced water oxidation taking place at a Mn cluster in this complex. We developed a set of tools for the study of PSII at XFELs, including a new liquid jet based on electrofocusing, an energy dispersive von Hamos X-ray emission spectrometer for the hard X-ray range and a high-throughput soft X-ray spectrometer based on a reflection zone plate. While our immediate focus is on PSII, the methods we describe here are applicable to a wide range of metalloenzymes. These experimental developments were complemented by a new software suite, cctbx.xfel. This software suite allows for near-real-time monitoring of the experimental parameters and detector signals and the detailed analysis of the diffraction and spectroscopy data collected by us at the Linac Coherent Light Source, taking into account the specific characteristics of data measured at an XFEL.     

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

Femtosecond crystallography of membrane proteins in the lipidic cubic phase

Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.     

自由电子激光在生物学中的应用

同步辐射的发展和应用已经极大的推动了自然科学包括生物学的巨大发展,其中结构生物学更是离不开X射线衍射分析,小角散射等。X射线自由电子激光(XFEL)相比同步辐射具有更高强度,完全相干等特点,被称为第四代光源。科学家已经利用XFEL实现了尺度约为1微米的蛋白质晶体的高分辨率结构解析,并且也实现了单颗粒的病毒的低分辨重构。未来,XFEL将会为生物学的发展打开一扇新的大门。     

Deducing fast electron density changes in randomly orientated uncrystallized biomolecules in a pump–probe experiment

We propose a method for deducing time-resolved structural changes in uncrystallized biomolecules in solution. The method relies on measuring the angular correlations of the intensities, when averaged over a large number of diffraction patterns from randomly oriented biomolecules in solution in a liquid solvent. The experiment is somewhat like a pump–probe version of an experiment on small angle X-ray scattering, except that the data expected by the algorithm are not just the radial variation of the averaged intensities. The differences of these correlation functions as measured from a photoexcited and dark structure enable the direct calculation of the difference electron density with a knowledge of only the dark structure. We exploit a linear relation we derive between the difference in these correlation functions and the difference electron density, applicable for small structural changes.     

Coherent convergent-beam time-resolved X-ray diffraction

The use of coherent X-ray lasers for structural biology allows the use of nanometre diameter X-ray beams with large beam divergence. Their application to the structure analysis of protein nanocrystals and single particles raises new challenges and opportunities. We discuss the form of these coherent convergent-beam (CCB) hard X-ray diffraction patterns and their potential use for time-resolved crystallography, normally achieved by Laue (polychromatic) diffraction, for which the monochromatic laser radiation of a free-electron X-ray laser is unsuitable. We discuss the possibility of obtaining single-shot, angle-integrated rocking curves from CCB patterns, and the dependence of the resulting patterns on the focused beam coordinate when the beam diameter is larger or smaller than a nanocrystal, or smaller than one unit cell. We show how structure factor phase information is provided at overlapping interfering orders and how a common phase origin between different shots may be obtained. Their use in refinement of the phase-sensitive intensity between overlapping orders is suggested.     

Potential of compact Compton sources in the medical field

The exceptional improvement of high power lasers and optical cavity finesses in the last fifteen years allows today the development of X-ray sources based on inverse Compton scattering. These compact sources will provide high intensity beams, with a tunable energy in the range 20–100 keV, that can be used in several application including material sciences, structural biology, cultural heritage research and preservation and medical or biomedical preclinical and clinical research. The access to these devices will be easier. Methods currently used only in synchrotron facilities will be available in dedicated work environment such as hospitals, laboratories or museums. Several machines are in design or construction phase, and aim at producing 10¹²–10¹⁴ ph/s. The ThomX machine is the most advanced project and has the potential to be used as the radiation source for biomedical searches, clinical imaging techniques or radiotherapy programs.     

7 ? resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump–probe experiments at subpicosecond time resolution.     

Effects of intracellular sodium and potassium iontophoresis on membrane potentials and resistances in toad urinary bladder

Conclusion The elastic X-ray/neutron diffraction techniques described in this review are currently capable of providing substantial information concerning the time-averaged structures and intermolecular ordering of molecular components within a dynamic membrane structure. In addition, the time resolution of the elastic X-ray diffraction technique, afforded by intense synchrotron and laser plasma X-ray sources, now permits this structural information to be obtained over a range of time scales from nanoseconds to milliseconds and upwards following an excitation of the membrane system. This time-averaged and time-resolved structural information may provide considerable insight into structure-function relationships in biological membranes and, especially when combined with structural information on the membrane proteins involved at atomic resolution, may provide this insight at the atomic level.     

Destruction‐and‐diffraction by X‐ray free‐electron laser

It is common knowledge that macromolecular crystals are damaged by the X‐rays they are exposed to during conventional data collection. One of the claims made about the crystallographic data collection now being collected using X‐ray free‐electron lasers (XFEL) is that they are unaffected by radiation damage. XFEL data sets are assembled by merging data obtained from a very large number of crystals, each of which is exposed to a single femtosecond pulse of radiation, the duration of which is so short that diffraction occurs before the damage done to the crystal has time to become manifest, i.e. “diffraction‐before‐destruction.” However, recent theoretical studies have shown that many of the elemental electronic processes that ultimately result in the destruction of such crystals occur during a single pulse. It is predicted that the amplitudes of atomic scattering factor could be reduced by as much as 75% within the first 5 femtoseconds of such pulses, and that different atoms will respond in different ways. Experimental evidence is provided here that these predictions are correct.     

High-resolution structure of viruses from random diffraction snapshots

The advent of the X-ray free-electron laser (XFEL) has made it possible to record diffraction snapshots of biological entities injected into the X-ray beam before the onset of radiation damage. Algorithmic means must then be used to determine the snapshot orientations and thence the three-dimensional structure of the object. Existing Bayesian approaches are limited in reconstruction resolution typically to 1/10 of the object diameter, with the computational expense increasing as the eighth power of the ratio of diameter to resolution. We present an approach capable of exploiting object symmetries to recover three-dimensional structure to high resolution, and thus reconstruct the structure of the satellite tobacco necrosis virus to atomic level. Our approach offers the highest reconstruction resolution for XFEL snapshots to date and provides a potentially powerful alternative route for analysis of data from crystalline and nano-crystalline objects.     

Unveiling the Timescale of the R-T Transition in Human Hemoglobin

Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place ∼ 2 μs after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-μs step observed with time-resolved optical spectroscopy corresponds to a small and localized structural change.     

Diffraction before destruction

X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.     

Rebuilding ships while at sea—Character individuality,homology, and evolutionary innovation

How novel traits originate in evolution is still one of the most perplexing questions in Evolutionary Biology. Building on a previous account of evolutionary innovation, I here propose that evolutionary novelties are those individualized characters that are not homologous to any characters in the ancestor. To clarify this definition, I here provide a detailed analysis of the concepts of “character individuality” and “homology” first, before addressing their role for our understanding of evolutionary innovation. I will argue (1) that functional as well as structural considerations are important for character individualization; and (2) that compositional (structural) and positional homology need to be clearly distinguished to properly describe the evolutionary transformations of hierarchically structured characters. My account will therefore integrate functional and structural perspectives and put forward a new multi-level view of character identity and transformation.     

Transmission electron microscopy as a tool for nanocrystal characterization pre- and post-injector

Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.     

Toward structure determination using membrane-protein nanocrystals and microcrystals

Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only 100 unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (～70fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-? wavelength, Bragg peaks were recorded to 8.5-? resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [94]. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.