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Femtosecond crystallography with an X-ray free-electron laser is used to analyse micrometre-sized protein crystals, generating a high-resolution structure of the protein without previous knowledge of what it looks like. Protein structures from smaller crystals X-ray crystallographers typically spend a great deal of time optimizing crystallization conditions to obtain the large, well-ordered crystals needed to generate high-quality data sets. It was shown recently that extremely short and intense pulses of X-rays from X-ray free-electron lasers can be used to obtain diffraction data on nano-to-micrometre-sized protein crystals before radiation damage to the crystal occurs. The hope is that this approach — called serial femtosecond crystallography — will produce structures of proteins and protein complexes that do not yield macroscopic, well-ordered crystals. One of the major limitations of serial femtosecond crystallography is that it has not been possible to solve the structure of a protein without prior knowledge of related, known structures. In this paper, the authors show how serial femtosecond crystallography with an X-ray free-electron laser can be used to experimentally solve the 'phase problem', generating a high-resolution structure of a protein without a priori knowledge of what the protein looks like. 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 1 , 2 , 3 , 4 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 1 , 2 , 3 , 4 , 5 . 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 1 , 2 , 3 , 4 , 6 , 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 6 , 7 , 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 1 , 2 , 8 .

There is a fundamental interest in studying photoinduced dynamics in nanoparticles and nanostructures as it provides insight into their mechanical and thermal properties out of equilibrium and during phase transitions. Nanoparticles can display significantly different properties from the bulk, which is due to the interplay between their size, morphology, crystallinity, defect concentration, and surface properties. Particularly interesting scenarios arise when nanoparticles undergo phase transitions, such as melting induced by an optical laser. Current theoretical evidence suggests that nanoparticles can undergo reversible nonhomogenous melting with the formation of a core-shell structure consisting of a liquid outer layer. To date, studies from ensembles of nanoparticles have tentatively suggested that such mechanisms are present. Here we demonstrate imaging transient melting and softening of the acoustic phonon modes of an individual gold nanocrystal, using an X-ray free electron laser. The results demonstrate that the transient melting is reversible and nonhomogenous, consistent with a core-shell model of melting. The results have implications for understanding transient processes in nanoparticles and determining their elastic properties as they undergo phase transitions. Significance Despite phase transitions, such as melting, being ubiquitous in nature, understanding what occurs at the nanoscale (such as in nanocrystals) has so far remained challenging. With ensemble studies of nanocrystals it is often difficult to discriminate between intrinsic size-dependent properties and effects due to sample size and shape dispersity. Here, using an X-ray free electron laser we image the reversible melting of an individual nanocrystal induced by an ultrashort laser. It is revealed that the melting occurs transiently, repeatably, and inhomogeneously. This is consistent with a core-shell model where the exterior is melted and a solid core remains. These findings reveal, unambiguously, that core-shell melting occurs, which has important implications for understanding nanoscale phenomena.

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.

The bright ultrafast pulses of X-ray Free-Electron Lasers allow investigation into the structure of matter under extreme conditions. We have used single pulses to ionize and probe water as it undergoes a phase transition from liquid to plasma. We report changes in the structure of liquid water on a femtosecond time scale when irradiated by single 6.86 keV X-ray pulses of more than 10⁶ J/cm². These observations are supported by simulations based on molecular dynamics and plasma dynamics of a water system that is rapidly ionized and driven out of equilibrium. This exotic ionic and disordered state with the density of a liquid is suggested to be structurally different from a neutral thermally disordered state.

The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this “probe-before-destroy” approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kβ ₁,₃ XES spectra of Mn ᴵᴵ and Mn ₂ᴵᴵᴵ,ᴵⱽ complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.

The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis. Serial femtosecond crystallography and the use of X-ray free-electron lasers (XFEL) promise to revolutionize structural biology. Here, the authors describe refinements that reduce the redundancy required to obtain quality XFEL data and report a 1.75-Å structure—not obtainable by synchrotron radiation—using less than 6,000 crystals.

We present a supervised deep neural network model for phase retrieval of coherent X-ray imaging and evaluate the performance. A supervised deep-learning-based approach requires a large amount of pre-training datasets. In most proposed models, the various experimental uncertainties are not considered when the input dataset, corresponding to the diffraction image in reciprocal space, is generated. We explore the performance of the deep neural network model, which is trained with an ideal quality of dataset, when it faces real-like corrupted diffraction images. We focus on three aspects of data qualities such as a detection dynamic range, a degree of coherence and noise level. The investigation shows that the deep neural network model is robust to a limited dynamic range and partially coherent X-ray illumination in comparison to the traditional phase retrieval, although it is more sensitive to the noise than the iteration-based method. This study suggests a baseline capability of the supervised deep neural network model for coherent X-ray imaging in preparation for the deployment to the laboratory where diffraction images are acquired.

We present a deep learning-based generative model for the enhancement of partially coherent diffractive images. In lensless coherent diffractive imaging, a highly coherent X-ray illumination is required to image an object at high resolution. Non-ideal experimental conditions result in a partially coherent X-ray illumination, lead to imperfections of coherent diffractive images recorded on a detector, and ultimately limit the capability of lensless coherent diffractive imaging. The previous approaches, relying on the coherence property of illumination, require preliminary experiments or expensive computations. In this article, we propose a generative adversarial network (GAN) model to enhance the visibility of fringes in partially coherent diffractive images. Unlike previous approaches, the model is trained to restore the latent sharp features from blurred input images without finding coherence properties of illumination. We demonstrate that the GAN model performs well with both coherent diffractive imaging and ptychography. It can be applied to a wide range of imaging techniques relying on phase retrieval of coherent diffraction patterns.

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli . The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. Diacylglycerol kinase is a small bacterial membrane-bound trimer that catalyses diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution.