The Ring-Closing Reaction of Cyclopentadiene Probed with Ultrafast X-ray Scattering.

The dynamics of cyclopentadiene (CP) following optical excitation at 243 nm was investigated by time-resolved pump-probe X-ray scattering using 16.2 keV X-rays at the Linac Coherent Light Source (LCLS). We present the first ultrafast structural evidence that the reaction leads directly to the formation of bicyclo[2.1.0]pentene (BP), a strained molecule with three- and four-membered rings. The bicyclic compound decays via a thermal backreaction to the vibrationally hot CP with a time constant of 21 ± 3 ps. A minor channel leads to ring-opened structures on a subpicosecond time scale.

Microstructure and crystal order during freezing of supercooled water drops.

Supercooled water droplets are widely used to study supercooled water1,2, ice nucleation3-5 and droplet freezing6-11. Their freezing in the atmosphere affects the dynamics and climate feedback of clouds12,13 and can accelerate cloud freezing through secondary ice production14-17. Droplet freezing occurs at several timescales and length scales14,18 and is sufficiently stochastic to make it unlikely that two frozen drops are identical. Here we use optical microscopy and X-ray laser diffraction to investigate the freezing of tens of thousands of water microdrops in vacuum after homogeneous ice nucleation around 234-235 K. On the basis of drop images, we developed a seven-stage model of freezing and used it to time the diffraction data. Diffraction from ice crystals showed that long-range crystalline order formed in less than 1 ms after freezing, whereas diffraction from the remaining liquid became similar to that from quasi-liquid layers on premelted ice19,20. The ice had a strained hexagonal crystal structure just after freezing, which is an early metastable state that probably precedes the formation of ice with stacking defects8,9,18. The techniques reported here could help determine the dynamics of freezing in other conditions, such as drop freezing in clouds, or help understand rapid solidification in other materials.

Transient vibration and product formation of photoexcited CS2 measured by time-resolved x-ray scattering.

We have observed details of the internal motion and dissociation channels in photoexcited carbon disulfide (CS2) using time-resolved x-ray scattering (TRXS). Photoexcitation of gas-phase CS2 with a 200 nm laser pulse launches oscillatory bending and stretching motion, leading to dissociation of atomic sulfur in under a picosecond. During the first 300 fs following excitation, we observe significant changes in the vibrational frequency as well as some dissociation of the C-S bond, leading to atomic sulfur in the both 1D and 3P states. Beyond 1400 fs, the dissociation is consistent with primarily 3P atomic sulfur dissociation. This channel-resolved measurement of the dissociation time is based on our analysis of the time-windowed dissociation radial velocity distribution, which is measured using the temporal Fourier transform of the TRXS data aided by a Hough transform that extracts the slopes of linear features in an image. The relative strength of the two dissociation channels reflects both their branching ratio and differences in the spread of their dissociation times. Measuring the time-resolved dissociation radial velocity distribution aids the resolution of discrepancies between models for dissociation proposed by prior photoelectron spectroscopy work.

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.

Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography.

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).

Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature.

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 Å resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 Å resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing.

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

Ultrafast X-ray scattering offers a structural view of excited-state charge transfer.

Intramolecular charge transfer and the associated changes in molecular structure in N,N'-dimethylpiperazine are tracked using femtosecond gas-phase X-ray scattering. The molecules are optically excited to the 3p state at 200 nm. Following rapid relaxation to the 3s state, distinct charge-localized and charge-delocalized species related by charge transfer are observed. The experiment determines the molecular structure of the two species, with the redistribution of electron density accounted for by a scattering correction factor. The initially dominant charge-localized state has a weakened carbon-carbon bond and reorients one methyl group compared with the ground state. Subsequent charge transfer to the charge-delocalized state elongates the carbon-carbon bond further, creating an extended 1.634 Å bond, and also reorients the second methyl group. At the same time, the bond lengths between the nitrogen and the ring-carbon atoms contract from an average of 1.505 to 1.465 Å. The experiment determines the overall charge transfer time constant for approaching the equilibrium between charge-localized and charge-delocalized species to 3.0 ps.

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

Unraveling the Unconventional Order of a High-Mobility Indacenodithiophene-Benzothiadiazole Copolymer.

A new class of donor-acceptor (D-A) copolymers found to produce high charge carrier mobilities competitive with amorphous silicon (>1 cm2 V-1 s-1) exhibit the puzzling microstructure of substantial local order, however lacking long-range order and crystallinity previously deemed necessary for achieving high mobility. Here, we demonstrate the application of low-dose transmission electron microscopy to image and quantify the nanoscale and mesoscale organization of an archetypal D-A copolymer across areas comparable to electronic devices (≈9 µm2). The local structure is spatially resolved by mapping the backbone (001) spacing reflection, revealing nanocrystallites of aligned polymer chains throughout nearly the entire film. Analysis of the nanoscale structure of its ordered domains suggests significant short- and medium-range order and preferential grain boundary orientations. Moreover, we provide insights into the rich, interconnected mesoscale organization of this new family of D-A copolymers by analysis of the local orientational spatial autocorrelations.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Observation of Seeded Mn Kß Stimulated X-Ray Emission Using Two-Color X-Ray Free-Electron Laser Pulses.

Kß x-ray emission spectroscopy is a powerful probe for electronic structure analysis of 3d transition metal systems and their ultrafast dynamics. Selectively enhancing specific spectral regions would increase this sensitivity and provide fundamentally new insights. Recently we reported the observation and analysis of Kα amplified spontaneous x-ray emission from Mn solutions using an x-ray free-electron laser to create the 1s core-hole population inversion [Kroll et al., Phys. Rev. Lett. 120, 133203 (2018)PRLTAO0031-900710.1103/PhysRevLett.120.133203]. To apply this new approach to the chemically more sensitive but much weaker Kß x-ray emission lines requires a mechanism to outcompete the dominant amplification of the Kα emission. Here we report the observation of seeded amplified Kß x-ray emission from a NaMnO_{4} solution using two colors of x-ray free-electron laser pulses, one to create the 1s core-hole population inversion and the other to seed the amplified Kß emission. Comparing the observed seeded amplified Kß emission signal with that from conventional Kß emission into the same solid angle, we obtain a signal enhancement of more than 10^{5}. Our findings are the first important step of enhancing and controlling the emission of selected final states of the Kß spectrum with applications in chemical and materials science.

Observation of the molecular response to light upon photoexcitation.

When a molecule interacts with light, its electrons can absorb energy from the electromagnetic field by rapidly rearranging their positions. This constitutes the first step of photochemical and photophysical processes that include primary events in human vision and photosynthesis. Here, we report the direct measurement of the initial redistribution of electron density when the molecule 1,3-cyclohexadiene (CHD) is optically excited. Our experiments exploit the intense, ultrashort hard x-ray pulses of the Linac Coherent Light Source (LCLS) to map the change in electron density using ultrafast x-ray scattering. The nature of the excited electronic state is identified with excellent spatial resolution and in good agreement with theoretical predictions. The excited state electron density distributions are thus amenable to direct experimental observation.

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

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 (λ ≈ 130 pm) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the Cornell-SLAC pixel array 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 be 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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Scattering off molecules far from equilibrium.

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.

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

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.

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

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.

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

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.