Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Crystallographic Studies of Rhodopsins: Structure and Dynamics.

Crystal structures have provided detailed insight in the architecture of rhodopsin photoreceptors. Of particular interest are the protein-chromophore interactions that govern the light-induced retinal isomerization and ultimately induce the large structural changes important for the various biological functions of the family. The reaction intermediates occurring along the rhodopsin photocycle have vastly differing lifetimes, from hundreds of femtoseconds to milliseconds. Detailed insight at high spatial and temporal resolution can be obtained by time-resolved crystallography using pump-probe approaches at X-ray free-electron lasers. Alternatively, cryotrapping approaches can be used. Both the approaches are described, including illumination and sample delivery. The importance of appropriate photoexcitation avoiding multiphoton absorption is stressed.

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

Effect of X-ray free-electron laser-induced shockwaves on haemoglobin microcrystals delivered in a liquid jet.

X-ray free-electron lasers (XFELs) enable obtaining novel insights in structural biology. The recently available MHz repetition rate XFELs allow full data sets to be collected in shorter time and can also decrease sample consumption. However, the microsecond spacing of MHz XFEL pulses raises new challenges, including possible sample damage induced by shock waves that are launched by preceding pulses in the sample-carrying jet. We explored this matter with an X-ray-pump/X-ray-probe experiment employing haemoglobin microcrystals transported via a liquid jet into the XFEL beam. Diffraction data were collected using a shock-wave-free single-pulse scheme as well as the dual-pulse pump-probe scheme. The latter, relative to the former, reveals significant degradation of crystal hit rate, diffraction resolution and data quality. Crystal structures extracted from the two data sets also differ. Since our pump-probe attributes were chosen to emulate EuXFEL operation at its 4.5 MHz maximum pulse rate, this prompts concern about such data collection.

Illumination guidelines for ultrafast pump-probe experiments by serial femtosecond crystallography.

Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.

MHz data collection of a microcrystalline mixture of different jack bean proteins.

We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.

Sample delivery for serial crystallography at free-electron lasers and synchrotrons.

The high peak brilliance and femtosecond pulse duration of X-ray free-electron lasers (XFELs) provide new scientific opportunities for experiments in physics, chemistry and biology. In structural biology, one of the major applications is serial femtosecond crystallography. The intense XFEL pulse results in the destruction of any exposed microcrystal, making serial data collection mandatory. This requires a high-throughput serial approach to sample delivery. To this end, a number of such sample-delivery techniques have been developed, some of which have been ported to synchrotron sources, where they allow convenient low-dose data collection at room temperature. Here, the current sample-delivery techniques used at XFEL and synchrotron sources are reviewed, with an emphasis on liquid injection and high-viscosity extrusion, including their application for time-resolved experiments. The challenges associated with sample delivery at megahertz repetition-rate XFELs are also outlined.

Crystallography on a chip - without the chip: sheet-on-sheet sandwich.

Crystallography chips are fixed-target supports consisting of a film (for example Kapton) or wafer (for example silicon) that is processed using semiconductor-microfabrication techniques to yield an array of wells or through-holes in which single microcrystals can be lodged for raster-scan probing. Although relatively expensive to fabricate, chips offer an efficient means of high-throughput sample presentation for serial diffraction data collection at synchrotron or X-ray free-electron laser (XFEL) sources. Truly efficient loading of a chip (one microcrystal per well and no wastage during loading) is nonetheless challenging. The wells or holes must match the microcrystal size of interest, requiring that a large stock of chips be maintained. Raster scanning requires special mechanical drives to step the chip rapidly and with micrometre precision from well to well. Here, a `chip-less' adaptation is described that essentially eliminates the challenges of loading and precision scanning, albeit with increased, yet still relatively frugal, sample usage. The device consists simply of two sheets of Mylar with the crystal solution sandwiched between them. This sheet-on-sheet (SOS) sandwich structure has been employed for serial femtosecond crystallography data collection with micrometre-sized crystals at an XFEL. The approach is also well suited to time-resolved pump-probe experiments, in particular for long time delays. The SOS sandwich enables measurements under XFEL beam conditions that would damage conventional chips, as documented here. The SOS sheets hermetically seal the sample, avoiding desiccation of the sample provided that the X-ray beam does not puncture the sheets. This is the case with a synchrotron beam but not with an XFEL beam. In the latter case, desiccation, setting radially outwards from each punched hole, sets lower limits on the speed and line spacing of the raster scan. It is shown that these constraints are easily accommodated.

Megahertz data collection from protein microcrystals at an X-ray free-electron laser.

X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10-120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a ~15 µm XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined.

Velocimetry of fast microscopic liquid jets by nanosecond dual-pulse laser illumination for megahertz X-ray free-electron lasers.

To conduct X-ray Free-Electron Laser (XFEL) measurements at megahertz (MHz) repetition rates, sample solution must be delivered in a micron-sized liquid free-jet moving at up to 100 m/s. This exceeds by over a factor of two the jet speeds measurable with current high-speed camera techniques. Accordingly we have developed and describe herein an alternative jet velocimetry based on dual-pulse nanosecond laser illumination. Three separate implementations are described, including a small laser-diode system that is inexpensive and highly portable. We have also developed and describe analysis techniques to automatically and rapidly extract jet speed from dual-pulse images.

Two-colour serial femtosecond crystallography dataset from gadoteridol-derivatized lysozyme for MAD phasing.

We provide a detailed description of a gadoteridol-derivatized lysozyme (gadolinium lysozyme) two-colour serial femtosecond crystallography (SFX) dataset for multiple wavelength anomalous dispersion (MAD) structure determination. The data was collected at the Spring-8 Angstrom Compact free-electron LAser (SACLA) facility using a two-colour double-pulse beam to record two diffraction patterns simultaneously in one diffraction image. Gadolinium lysozyme was chosen as a well-established model system that has a very strong anomalous signal. Diffraction patterns from gadolinium lysozyme microcrystals were recorded to a resolution of 1.9 Å in both colours. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development.

Multi-wavelength anomalous diffraction de novo phasing using a two-colour X-ray free-electron laser with wide tunability.

Serial femtosecond crystallography at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems prone to radiation damage. However, de novo structure determination, i.e., without prior structural knowledge, is complicated by the inherent inaccuracy of serial femtosecond crystallography data. By its very nature, serial femtosecond crystallography data collection entails shot-to-shot fluctuations in X-ray wavelength and intensity as well as variations in crystal size and quality that must be averaged out. Hence, to obtain accurate diffraction intensities for de novo phasing, large numbers of diffraction patterns are required, and, concomitantly large volumes of sample and long X-ray free-electron laser beamtimes. Here we show that serial femtosecond crystallography data collected using simultaneous two-colour X-ray free-electron laser pulses can be used for multiple wavelength anomalous dispersion phasing. The phase angle determination is significantly more accurate than for single-colour phasing. We anticipate that two-colour multiple wavelength anomalous dispersion phasing will enhance structure determination of difficult-to-phase proteins at X-ray free-electron lasers.

Viscous hydrophilic injection matrices for serial crystallography.

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates - gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro-gels as viscous injection matrices is described, namely sodium carb-oxy-methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.