Understanding structure at the atomic scale is fundamental for understanding the functioning and the development of materials with improved properties. Compared with other probes providing atomic resolution, electrons offer the strongest interaction in combination with minimal radiation damage, which makes them an ideal tool for investigating very small and radiation-sensitive samples [Henderson (1995), Q. Rev. Biophys. 28, 171-193]. However, these benefits are often offset by the laborious preparation of nanometre-sized samples that are not visible using a light microscope, and the fact that experiments are largely restricted to ultra-high vacuum [Duyvesteyn et al. (2018), Proc. Natl Acad. Sci. USA 115, 9569-9573; Gruene et al. (2021), Nat. Rev. Chem. 5, 660-668]. Here, we report the successful implementation of MeV electron diffraction for ab initio 3D structure determination of the quasi-2D material muscovite and the quantum material 1T-TaS2 at atomic resolution. By employing ultrashort electron pulses from the REGAE (Relativistic electron gun for atomic exploration) accelerator, we obtained high-quality diffraction datasets suitable for structural refinements based on dynamical scattering theory, enabling precise localization of even hydrogen atoms. The increased penetration depth of MeV electrons significantly expands the applicable thickness range of samples, overcoming previous restrictions associated with traditional electron diffraction. These findings establish MeV electron diffraction as a viable approach for investigating a broad range of materials, including nanostructures and radiation-sensitive compounds, and open up new opportunities for in situ and time-resolved experiments [Chao et al. (2023), Chem. Rev. 123, 8347-8394; Filippetto et al. (2022), Rev. Mod. Phys. 94, 045004].

Molecular and polymeric crystals show a wide range of functional properties that arise from the interplay between the atomic-scale structure of their constituent molecules and the organization of these molecules within the crystal at macroscopic length scales. X-ray diffraction can provide structural information at these disparate length scales, but usually only through experiments that address one or the other of molecular (or unit-cell) structure versus crystal structure. Consequently, the accuracy of determined molecular or polymer structures may be limited by unaccounted for crystal inhomogeneities and the characterization of crystalline materials might not reveal the underlying causes of crystal morphology. Here we introduce X-ray convergent-beam diffraction to obtain spatially resolved structural information from crystals by projection topographic imaging. Using highly focusing X-ray multilayer Laue lenses, we show that Bragg reflections can be mapped into tomographic images of the crystal, for the characterization of strain and defects at high resolution. We demonstrate how the crystal morphology obtained this way can be accounted for when determining structure factors as a function of position in the crystal. The approach may assist in studies such as diffusion and binding in metal-organic framework structures, protein-drug binding, crystal growth, and the mechanical responses of photo-reactive or thermally driven dynamic crystals.

Structural insights into protein-ligand interactions are essential for advancing drug development, with macromolecular X-ray crystallography being a cornerstone technique. Commonly X-ray data collection is conducted at cryogenic temperatures to mitigate radiation damage effects. However, this can introduce artifacts not only in the protein conformation but also in protein-ligand interactions. Recent studies highlight the advantages of room-temperature (RT) crystallography in capturing relevant states much closer to physiological temperatures. We have advanced fixed-target serial crystallography to enable high-throughput fragment screening at RT. Here we systematically compare RT fragment screening of the Fosfomycin-resistance protein A from Klebsiella pneumoniae (FosAKP), an enzyme involved in antibiotic resistance, with conventional single crystal data collection at cryogenic temperature (cryo). With RT serial crystallography we achieve resolutions comparable to cryogenic methods and identify a previously unobserved conformational state of the active site, offering additional starting points for drug design. For ligands identified in both screens, temperature does not have an influence on the binding mode of the ligand. But overall, we observe more binders at cryo, both at physiologically relevant and non-relevant sites. With the potential for further automation, RT screening with serial crystallography can advance drug development pipelines by making undiscovered conformations of proteins accessible.

Understanding structure at the atomic scale is fundamental for the development of materials with improved properties. Compared to other probes providing atomic resolution, electrons offer the strongest interaction in combination with minimal radiation damage. Here, we report the successful implementation of MeV electron diffraction for ab initio 3D structure determination at atomic resolution. Using ultrashort electron pulses from the REGAE accelerator, we obtained high-quality diffraction data from muscovite and $1T-TaS_2$, enabling structure refinements according to the dynamical scattering theory and the accurate determination of hydrogen atom positions. The increased penetration depth of MeV electrons allows for structure determination from samples significantly thicker than those typically applicable in electron diffraction. These findings establish MeV electron diffraction as a viable approach for investigating a broad range of materials, including nanostructures and radiation-sensitive compounds, and open up new opportunities for in-situ and time-resolved experiments.

We demonstrate that x-ray fluorescence emission, which cannot maintain a stationary interference pattern, can be used to obtain images of structures by recording photon-photon correlations in the manner of the stellar intensity interferometry of Hanbury Brown and Twiss. This is achieved utilizing femtosecond-duration pulses of a hard x-ray free-electron laser to generate the emission in exposures comparable to the coherence time of the fluorescence. Iterative phasing of the photon correlation map generated a model-free real-space image of the structure of the emitters. Since fluorescence can dominate coherent scattering, this may enable imaging uncrystallised macromolecules.

Structural insight into nano-objects down to the atomic scale is one of the most important prerequisites to understand the properties of functional materials and will ultimately permit one to relate the size and shape of nanoparticles to their catalytic activity. We elucidate the potential of extracting structural information about a small ensemble of nanoparticles that are semiregularly arranged on a periodic array from coherent x-ray Bragg diffraction. The observed fringe pattern in the Pt(111) Bragg peak obviously originates from the mutual interference of the Bragg scattered wave field from individual nanoparticles in the nanoarray. Despite the absence of a symmetry center in the Bragg peak of the nanoarray, we identify the most prominent in-plane spatial frequencies of the latter by applying a Patterson map analysis to the Bragg peak superstructure. Integration along the in-plane reciprocal space direction over the relevant in-plane regions of interest results in Laue oscillations that arise from nanoparticle sets of similar heights in real space. A one-to-one comparison with real-space microscopic information obtained from scanning electron microscopy and atomic force microscopy suggests potential nanoparticle subsets as the origin for the x-ray intensity in these regions of interest by the good agreement in their height and direction-dependent in-plane interparticle distances, as also further supported by simulations. Nanoparticle arrays with well-defined tunable sizes and lateral distances may serve in the future to track structural changes in, e.g., sizes, relative positions, and tilts of smallest' catalysis-relevant nanoparticles during operando heterogeneous catalysis experiments in the 10-nm-size regime.

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics.

Here we present the crystal structure of SARS-CoV-2 main protease (M pro ) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of M pro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of M pro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by M pro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by M pro , followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychography-enhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix.

Nanolipoprotein particles (NLPs), also called "nanodiscs", are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via HDL and LDL maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics and develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar in characteristics to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

We report on a method of fabricating lanthanum hexaboride (LaB6) cold field emission tips with sub-100-nm apices by using a combination of electrochemical etching and focused ion beam milling. The primary advantage of combining the two methods is rapid fabrication while maintaining reproducibility. The LaB6 tips have low work functions and high mechanical stabilities and are chemically inert to residual gases. Field emission characterization was performed on three tips, with apex sizes of 15, 85, and 80 nm yielding 10 nA cold field emission currents at 0.76, 3.9, and 3.6 kV extraction potentials, respectively. All three tips showed excellent emission current stability for periods exceeding 30 min in a 5 × 10−9 mbar vacuum.

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.

Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a doubleflow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double-flow focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.