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Supercooled water droplets are widely used to study supercooled water 1 , 2 , ice nucleation 3 – 5 and droplet freezing 6 – 11 . Their freezing in the atmosphere affects the dynamics and climate feedback of clouds 12 , 13 and can accelerate cloud freezing through secondary ice production 14 – 17 . Droplet freezing occurs at several timescales and length scales 14 , 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 ice 19 , 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 defects 8 , 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. Optical microscopy and X-ray diffraction are used to study the freezing of water droplets in vacuum, leading to the development of a seven-stage model of freezing and the mapping of ice structures and crystal order.

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. Quantum coherence and dephasing in molecular motions determine the behaviour of many chemical reactions and are the fundamental basis for the concept of coherent control. Now, ultrafast X-ray scattering combined with a detailed structural determination analysis precisely measures the coherent vibrational motions of a polyatomic organic molecule following photoexcitation.

Cytochrome c oxidase (C c O) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the C c O reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable O H state, and a reductive phase, in which O H is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if O H is allowed to relax to the resting oxidized state (O), a redox equivalent to O H , its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a 3 iron and Cu B in the active site of the O state, like those in the O H state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O H , where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of C c O. Using resonance Raman spectroscopy and serial femtosecond X-ray crystallography, the authors show the heme a 3 iron and Cu B in the resting oxidized form of Cytochrome c Oxidase are coordinated by a hydroxide ion and a water molecule, respectively.

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 .

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils. A short segment of α-synuclein called NACore (residues 68–78) is responsible for the formation of amyloid aggregates responsible for cytotoxicity in Parkinson disease; here the nanocrystal structure of this invisible-to-optical-microscopy segment is determined using micro-electron diffraction, offering insight into its function and simultaneously demonstrating the first use of micro-electron diffraction to solve a previously unknown protein structure. Structure of toxic α-synuclein The small protein α-synuclein (α-syn) relies on a short segment called NACore (residues 68–78) to form amyloid aggregates responsible for cytotoxicity in Parkinson's disease, but structural information about it has been lacking. Using the frontier-method of micro-electron diffraction (MicroED), David Eisenberg and collaborators have now determined the structure of NACore nanocrystals too small to even be visible by light microscopy. They observe a structure similar to that of toxic fibrils formed by full-length α-syn. The 1.4Å resolution of this structure is the highest achieved by any cryo-EM method to date, and the work shows, for the first time, that MicroED can be used to solve previously unknown protein structures.

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.

The Linac Coherent Light Source (LCLS) has significantly impacted the field of biology by providing advanced capabilities for probing the structure and dynamics of biological molecules with high precision. The ultrashort coherent X-ray pulses from the LCLS have enabled ultrafast, time-resolved, serial femtosecond crystallography that is inaccessible at conventional synchrotron light sources. Since the facility's founding, scientists have captured detailed insights into biological processes at atomic resolution and fundamental timescales. The ability to observe these processes in real time and under conditions closely resembling their natural state is transforming our approach to studying biochemical mechanisms and developing new medical and energy applications. This work recounts some of the history of the LCLS, advances in biological research enabled by the LCLS, key biological areas that have been impacted and how the LCLS has helped to unravel complex biological phenomena in these fields.

X-ray scattering is typically used as a weak linear atomic-scale probe of matter. At high intensities, such as produced at free-electron lasers, nonlinearities can become important, and the probe may no longer be considered weak. Here we report the observation of one of the most fundamental nonlinear X-ray–matter interactions: the concerted nonlinear Compton scattering of two identical hard X-ray photons producing a single higher-energy photon. The X-ray intensity reached 4 × 10 20  W cm −2 , corresponding to an electric field well above the atomic unit of strength and within almost four orders of magnitude of the quantum-electrodynamic critical field. We measure a signal from solid beryllium that scales quadratically in intensity, consistent with simultaneous non-resonant two-photon scattering from nearly-free electrons. The high-energy photons show an anomalously large redshift that is incompatible with a free-electron approximation for the ground-state electron distribution, suggesting an enhanced nonlinearity for scattering at large momentum transfer. Radiation–matter interactions can become highly nonlinear when using high-intensity X-ray free-electron lasers. Under such conditions, it is shown that nonlinear Compton scattering has an anomalous redshift, whose origin remains unclear.

Cytochrome c oxidase (CcO) 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 CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a₃ iron atom is in a ferryl (Fe4+ = O2−) configuration, and heme a and CuB are oxidized while CuA 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.

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.