Showing papers by "Sébastien Boutet published in 2019"

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Abstract: 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.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Abstract: Bacteriorhodopsin (bR) is a light-driven proton pump. We use 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 allow identifying a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multi-photon 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.

Ultrafast X-ray scattering reveals vibrational coherence following Rydberg excitation.

Abstract: The coherence and dephasing of vibrational motions of molecules constitute an integral part of chemical dynamics, influence material properties and underpin schemes to control chemical reactions. Considerable progress has been made in understanding vibrational coherence through spectroscopic measurements, but precise, direct measurement of the structure of a vibrating excited-state polyatomic organic molecule has remained unworkable. Here, we measure the time-evolving molecular structure of optically excited N-methylmorpholine through scattering with ultrashort X-ray pulses. The scattering signals are corrected for the differences in electron density in the excited electronic state of the molecule in comparison to the ground state. The experiment maps the evolution of the molecular geometry with femtosecond resolution, showing coherent motion that survives electronic relaxation and seems to persist for longer than previously seen using other methods.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Abstract: Cytochrome c oxidase (C c O) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine C c O. It is assigned to the P R -intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a 3 iron atom is in a ferryl (Fe 4+ = O 2− ) configuration, and heme a and Cu B are oxidized while Cu A is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis.

Abstract: How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source.

Abstract: The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

A deep UV trigger for ground-state ring-opening dynamics of 1,3-cyclohexadiene.

Abstract: We explore the photo-induced kinetics of 1,3-cyclohexadiene upon excitation at 200 nm to the 3p state by ultrafast time-resolved, gas-phase x-ray scattering using the Linac Coherent Light Source. Analysis of the scattering anisotropy reveals that the excitation leads to the 3px and 3py Rydberg electronic states, which relax to the ground state with a time constant of 208 ± 11 fs. In contrast to the well-studied 266 nm excitation, at 200 nm the majority of the molecules (76 ± 3%) relax to vibrationally hot cyclohexadiene in the ground electronic state. A subsequent reaction on the ground electronic state surface leads from the hot cyclohexadiene to 1,3,5-hexatriene, with rates for the forward and backward reactions of 174 ± 13 and 355 ± 45 ps, respectively. The scattering pattern of the final hexatriene product reveals a thermal distribution of rotamers about the carbon-carbon single bonds.

Simplicity Beneath Complexity: Counting Molecular Electrons Reveals Transients and Kinetics of Photodissociation Reactions

Abstract: Time-resolved pump-probe gas-phase X-ray scattering signals, extrapolated to zero momentum transfer, provide a measure of the number of electrons in a system, an effect that arises from the coherent addition of elastic scattering from the electrons. This allows to identify reactive transients and determine the chemical reaction kinetics without the need for extensive scattering simulations or complicated inversion of scattering data. We examine the photodissociation reaction of trimethylamine and identify two reaction paths upon excitation to the 3p state at 200 nm: a fast dissociation path out of the 3p state to the dimethyl amine radical (16.6±1.2 %) and a slower dissociation via internal conversion to the 3s state (83.4±1.2 %). The time constants for the two reactions are 640±130 fs and 74±6 ps, respectively. Additionally, it is found that the transient dimethyl amine radical has a N-C bond length of 1.45±0.02 A and a C-N-C bond angle of 118°±4°.

Coherent diffractive imaging of microtubules using an X-ray laser.

Abstract: X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.

3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating.

Abstract: The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

Scattering off molecules far from equilibrium

Abstract: Pump-probe gas phase X-ray scattering experiments, enabled by the development of X-ray free electron lasers, have advanced to reveal scattering patterns of molecules far from their equilibrium geometry. While dynamic displacements reflecting the motion of wavepackets can probe deeply into the reaction dynamics, in many systems, the thermal excitation embedded in the molecules upon optical excitation and energy randomization can create systems that encompass structures far from the ground state geometry. For polyatomic molecular systems, large amplitude vibrational motions are associated with anharmonicity and shifts of interatomic distances, making analytical solutions using traditional harmonic approximations inapplicable. More generally, the interatomic distances in a polyatomic molecule are not independent and the traditional equations commonly used to interpret the data may give unphysical results. Here, we introduce a novel method based on molecular dynamic trajectories and illustrate it on two examples of hot, vibrating molecules at thermal equilibrium. When excited at 200 nm, 1,3-cyclohexadiene (CHD) relaxes on a subpicosecond time scale back to the reactant molecule, the dominant pathway, and to various forms of 1,3,5-hexatriene (HT). With internal energies of about 6 eV, the energy thermalizes quickly, leading to structure distributions that deviate significantly from their vibrationless equilibrium. The experimental and theoretical results are in excellent agreement and reveal that a significant contribution to the scattering signal arises from transition state structures near the inversion barrier of CHD. In HT, our analysis clarifies that previous inconsistent structural parameters determined by electron diffraction were artifacts that might have resulted from the use of inapplicable analytical equations.

Performance of ePix10K, a high dynamic range, gain auto-ranging pixel detector for FELs

Abstract: ePix10K is a hybrid pixel detector developed at SLAC for demanding free-electron laser (FEL) applications, providing an ultrahigh dynamic range (245 eV to 88 MeV) through gain auto-ranging. It has three gain modes (high, medium and low) and two auto-ranging modes (high-to-low and medium-to-low). The first ePix10K cameras are built around modules consisting of a sensor flip-chip bonded to 4 ASICs, resulting in 352 × 384 pixels of 100 µm x 100 µm each. We present results from extensive testing of three ePix10K cameras with FEL beams at LCLS, resulting in a measured noise floor of 245 eV rms, or 67 e− equivalent noise charge (ENC), and a range of 11 000 photons at 8 keV. We demonstrate the linearity of the response in various gain combinations: fixed high, fixed medium, fixed low, auto-ranging high to low, and auto-ranging medium-to-low, while maintaining a low noise (well within the counting statistics), a very low cross-talk, perfect saturation response at fluxes up to 900 times the maximum range, and acquisition rates of up to 480 Hz. Finally, we present examples of high dynamic range x-ray imaging spanning more than 4 orders of magnitude dynamic range (from a single photon to 11 000 photons/pixel/pulse at 8 keV). Achieving this high performance with only one auto-ranging switch leads to relatively simple calibration and reconstruction procedures. The low noise levels allow usage with long integration times at non-FEL sources. ePix10K cameras leverage the advantages of hybrid pixel detectors with high production yield and good availability, minimize development complexity through sharing the hardware, software and DAQ development with all other versions of ePix cameras, while providing an upgrade path to 5 kHz, 25 kHz and 100 kHz in three steps over the next few years, matching the LCLS-II requirements.

Performance of ePix10K, a high dynamic range, gain auto-ranging pixel detector for FELs.

Abstract: ePix10K is a hybrid pixel detector developed at SLAC for demanding free-electron laser (FEL) applications, providing an ultrahigh dynamic range (245 eV to 88 MeV) through gain auto-ranging. It has three gain modes (high, medium and low) and two auto-ranging modes (high-to-low and medium-to-low). The first ePix10K cameras are built around modules consisting of a sensor flip-chip bonded to 4 ASICs, resulting in 352x384 pixels of 100 $\mu$m x 100 $\mu$m each. We present results from extensive testing of three ePix10K cameras with FEL beams at LCLS, resulting in a measured noise floor of 245 eV rms, or 67 e$^-$ equivalent noise charge (ENC), and a range of 11000 photons at 8 keV. We demonstrate the linearity of the response in various gain combinations: fixed high, fixed medium, fixed low, auto-ranging high to low, and auto-ranging medium-to-low, while maintaining a low noise (well within the counting statistics), a very low cross-talk, perfect saturation response at fluxes up to 900 times the maximum range, and acquisition rates of up to 480 Hz. Finally, we present examples of high dynamic range x-ray imaging spanning more than 4 orders of magnitude dynamic range (from a single photon to 11000 photons/pixel/pulse at 8 keV). Achieving this high performance with only one auto-ranging switch leads to relatively simple calibration and reconstruction procedures. The low noise levels allow usage with long integration times at non-FEL sources. ePix10K cameras leverage the advantages of hybrid pixel detectors with high production yield and good availability, minimize development complexity through sharing the hardware, software and DAQ development with all other versions of ePix cameras, while providing an upgrade path to 5 kHz, 25 kHz and 100 kHz in three steps over the next few years, matching the LCLS-II requirements.

Generation of high-intensity ultrasound through shock propagation in liquid jets

Abstract: An investigation shows how single shock waves, produced in water jets by x-ray laser pulses, propagate and become ultrasonic shock trains through oblique shock reflections and cavitation. These trains are one of the loudest sounds ever made in liquid water and may damage samples studied with x-ray lasers.

X-ray diffractive imaging of controlled gas-phase molecules: Toward imaging of dynamics in the molecular frame

Abstract: We report experimental results on the diffractive imaging of three-dimensionally aligned 2,5-diiodothiophene molecules. The molecules were aligned by chirped near-infrared laser pulses, and their structure was probed at a photon energy of 9.5 keV ($\lambda\approx130 \text{pm}$) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the CSPAD detector and a two-dimensional diffraction pattern of the equilibrium structure of 2,5-diiodothiophene was recorded. The retrieved distance between the two iodine atoms agrees with the quantum-chemically calculated molecular structure to within 5 %. The experimental approach allows for the imaging of intrinsic molecular dynamics in the molecular frame, albeit this requires more experimental data which should be readily available at upcoming high-repetition-rate facilities.

X-ray emission spectroscopy at X-ray free electron lasers: limits to observation of the classical spectroscopic response for electronic structure analysis

Abstract: X-ray free electron lasers (XFELs) provide ultrashort intense X-ray pulses suitable to probe electron dynamics but can also induce a multitude of nonlinear excitation processes. These affect spectroscopic measurements and interpretation, particularly for upcoming brighter XFELs. Here we identify and discuss the limits to observing classical spectroscopy, where only one photon is absorbed per atom for a Mn2+ in a light element (O, C, H) environment. X-ray emission spectroscopy (XES) with different incident photon energies, pulse intensities, and pulse durations is presented. A rate equation model based on sequential ionization and relaxation events is used to calculate populations of multiply ionized states during a single pulse and to explain the observed X-ray induced spectral lines shifts. This model provides easy estimation of spectral shifts, which is essential for experimental designs at XFELs and illustrates that shorter X-ray pulses will not overcome sequential ionization but can reduce electron ca...

Nanofocus characterization at the Coherent X-ray Imaging instrument using 2D single grating interferometry

Abstract: In this work we present the application of a 2D single grating wavefront sensor to align and characterize the 100 nm focus at the Coherent X-ray Imaging (CXI) endstation at the Linac Coherent Light Source (LCLS). The results agree well with a model of the system, indicating that the mirrors perform as designed when alignment is optimized. In addition, a comparison with the imprint technique confirms the validity of the results, which showed that wavefront-based alignment resulted in negligible astigmatism. Analysis of the retrieved focus profile indicates that intensities <1021 W=cm2 are achievable with currently available LCLS beam parameters and optimal mirror alignment.

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Abstract: Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.