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.

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.

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.

Current status and future opportunities for serial crystallography at MAX IV Laboratory.

Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at  MAX IV Laboratory in Lund, Sweden, are described, together with case studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.

BioMAX - the first macromolecular crystallography beamline at MAX IV Laboratory.

BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 µm × 5 µm using the bendable focusing mirrors. The beam is tunable in the energy range 5-25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 µm × 1 µm beam focus and a flux up to 1015 photons s-1 with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.

De novo protein crystal structure determination from X-ray free-electron laser data.

The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized crystals in a 'diffraction-before-destruction' approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins.

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser.

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

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.

Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser.

We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon.

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical ß-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical ß-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient ß-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

Femtosecond X-ray protein nanocrystallography.

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 µm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

Single mimivirus particles intercepted and imaged with an X-ray laser.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

Protein crystal structure obtained at 2.9 Å resolution from injecting bacterial cells into an X-ray free-electron laser beam.

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.

Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams.

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.

Lipidic phase membrane protein serial femtosecond crystallography.

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

Characterization and use of the spent beam for serial operation of LCLS.

X-ray free-electron laser sources such as the Linac Coherent Light Source offer very exciting possibilities for unique research. However, beam time at such facilities is very limited and in high demand. This has led to significant efforts towards beam multiplexing of various forms. One such effort involves re-using the so-called spent beam that passes through the hole in an area detector after a weak interaction with a primary sample. This beam can be refocused into a secondary interaction region and used for a second, independent experiment operating in series. The beam profile of this refocused beam was characterized for a particular experimental geometry at the Coherent X-ray Imaging instrument at LCLS. A demonstration of this multiplexing capability was performed with two simultaneous serial femtosecond crystallography experiments, both yielding interpretable data of sufficient quality to produce electron density maps.

Effects of self-seeding and crystal post-selection on the quality of Monte Carlo-integrated SFX data.

Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected in this way are then combined in a scheme called Monte Carlo integration, which provides the full diffraction intensities. However, apart from having to perform this merging, the Monte Carlo integration must also average out all variations in crystal quality, crystal size, X-ray beam properties and other factors, necessitating data collection from thousands of crystals. Because the pulses provided by FELs running in the typical self-amplified spontaneous emission (SASE) mode of operation have very irregular, spiky spectra that vary strongly from pulse to pulse, it has been suggested that this is an important source of variation contributing to inaccuracies in the intensities, and that, by using monochromatic pulses produced through a process called self-seeding, fewer images might be needed for Monte Carlo integration to converge, resulting in more accurate data. This paper reports the results of two experiments performed at the Linac Coherent Light Source in which data collected in both SASE and self-seeded mode were compared. Importantly, no improvement attributable to the use of self-seeding was detected. In addition, other possible sources of variation that affect SFX data quality were investigated, such as crystal-to-crystal variations reflected in the unit-cell parameters; however, these factors were found to have no influence on data quality either. Possibly, there is another source of variation as yet undetected that affects SFX data quality much more than any of the factors investigated here.

Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase.

The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.