3D electron diffraction (3D ED) has undergone impressive development in the last decade. However, its accuracy and reproducibility have never been tested, up to now, in different laboratories on the same batch of samples. This paper reports a round robin on three test structures, two inorganic and one organic, solved and refined with 3D ED in seven different laboratories employing different transmission electron microscopes, with different acceleration voltages, different methodologies and different detectors. The results of the round robin show a remarkable accuracy of the technique that, in the case of kinematical refinement, is around 0.05 Å error on atomic positions for the inorganic samples and 0.15 Å for the beam-sensitive organic crystal. Dynamical refinement further improves the accuracy. The analysis of diverse samples and numerous data sets again confirms that dynamical refinement is a well established procedure, significantly reducing the refinement R factors, improving the accuracy of the structure models in most cases, and providing fine structural details, such as hydrogen-atom positions and the absolute structure, for both inorganic and organic samples.

The amino sugar N- acetylglucosamine (GlcNAc) plays a central role in primary metabolism and is a key signaling molecule for the onset of morphological and chemical differentiation of Streptomyces . The global nutrient-sensory regulator DasR acts as the gatekeeper of development in streptomycetes, and its activity is modulated by aminosugar phosphates. Here, we report the discovery of a novel pathway in aminosugar metabolism that governs GlcNAc sensing. GlcNAc-6P is converted into a toxic metabolite via two new enzyme functions, namely dehydration of N -acetylglucosamine-6-phosphate by NagS to form 6P-Chromogen I, a reaction that has not yet been described in the textbooks, and its subsequent deacetylation by NagA producing a cytotoxic structural analogue of ribose. The latter reveals an unexpected promiscuous activity for GlcNAc-6P deacetylase NagA. The crystal structures of NagS apoenzyme and NagS in complex with its substrate GlcNAc-6P or its inhibitor 6-phosphogluconate were resolved at 2.3 Å, 2.6 Å, and 1.7 Å resolution, respectively. Detailed in vivo and in vitro studies resolved the key residues of the NagS catalytic site. Thus, we have uncovered a novel pathway in aminosugar metabolism that sheds new light on nutrient-mediated control of development and antibiotic production in Streptomyces . ### Competing Interest Statement The authors have declared no competing interest.

Deep learning techniques can recognize complex patterns in noisy, multidimensional data. In recent years, researchers have started to explore the potential of deep learning in the field of structural biology, including protein crystallography. This field has some significant challenges, in particular producing high-quality and well ordered protein crystals. Additionally, collecting diffraction data with high completeness and quality, and determining and refining protein structures can be problematic. Protein crystallographic data are often high-dimensional, noisy and incomplete. Deep learning algorithms can extract relevant features from these data and learn to recognize patterns, which can improve the success rate of crystallization and the quality of crystal structures. This paper reviews progress in this field.

IntroductionProteins that adopt multiple conformations pose significant challenges in structural biology research and pharmaceutical development, as structure determination via single particle cryo-electron microscopy (cryo-EM) is often impeded by data heterogeneity. In this context, the enhanced signal-to-noise ratio of single molecule cryo-electron diffraction (simED) offers a promising alternative. However, a significant challenge in diffraction methods is the loss of phase information, which is crucial for accurate structure determination.MethodsHere, we present DiffraGAN, a conditional generative adversarial network (cGAN) that estimates the missing phases at high resolution from a combination of single particle high-resolution diffraction data and low-resolution image data.ResultsFor simulated datasets, DiffraGAN allows effectively determining protein structures at atomic resolution from diffraction patterns and noisy low-resolution images.DiscussionOur findings suggest that combining single particle cryo-electron diffraction with advanced generative modeling, as in DiffraGAN, could revolutionize the way protein structures are determined, offering an alternative and complementary approach to existing methods.

The mitochondrial Lon protease (LonP1) regulates mitochondrial health by removing redundant proteins from the mitochondrial matrix. We determined LonP1 in eight nucleotide-dependent conformational states by cryoelectron microscopy (cryo-EM). The flexible assembly of N-terminal domains had 3-fold symmetry, and its orientation depended on the conformational state. We show that a conserved structural motif around T803 with a high similarity to the trypsin catalytic triad is essential for proteolysis. We show that LonP1 is not regulated by redox potential, despite the presence of two conserved cysteines at disulfide-bonding distance in its unfoldase core. Our data indicate how sequential ATP hydrolysis controls substrate protein translocation in a 6-fold binding change mechanism. Substrate protein translocation, rather than ATP hydrolysis, is a rate-limiting step, suggesting that LonP1 is a Brownian ratchet with ATP hydrolysis preventing translocation reversal. 3-fold rocking motions of the flexible N-domain assembly may assist thermal unfolding of the substrate protein.

Amyloid-β peptide (Aβ) oligomers are pathogenic species of amyloid aggregates in Alzheimer's disease. Like certain protein toxins, Aβ oligomers permeabilize cellular membranes, presumably through a pore formation mechanism. Owing to their structural and stoichiometric heterogeneity, the structure of these pores remains to be characterized. We studied a functional Aβ42-pore equivalent, created by fusing Aβ42 to the oligomerizing, soluble domain of the α-hemolysin (αHL) toxin. Our data reveal Aβ42-αHL oligomers to share major structural, functional, and biological properties with wild-type Aβ42-pores. Single-particle cryo-EM analysis of Aβ42-αHL oligomers (with an overall 3.3 Å resolution) reveals the Aβ42-pore region to be intrinsically flexible. The Aβ42-αHL oligomers will allow many of the features of the wild-type amyloid oligomers to be studied that cannot be otherwise, and may be a highly specific antigen for the development of immuno-base diagnostics and therapies.

Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.

In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium Streptomyces, cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here, we show that various amino acid (aa) substitutions in the highly conserved SsgB protein result in ectopically placed septa that sever spores diagonally or along the long axis, perpendicular to the division plane. Fluorescence microscopy revealed that between 3.3% and 9.8% of the spores of strains expressing SsgB E120 variants were severed ectopically. Biochemical analysis of SsgB variant E120G revealed that its interaction with FtsZ had been maintained. The crystal structure of Streptomyces coelicolor SsgB was resolved and the key residues were mapped on the structure. Notably, residue substitutions (V115G, G118V, E120G) that are associated with septum misplacement localize in the α2–α3 loop region that links the final helix and the rest of the protein. Structural analyses and molecular simulation revealed that these residues are essential for maintaining the proper angle of helix α3. Our data suggest that besides altering FtsZ, aa substitutions in the FtsZ-recruiting protein SsgB also lead to diagonally or longitudinally divided cells in Streptomyces.

It is a hot topic to improve efficiency and decrease toxicity of gene transfection reagents. The extracellular nanovesicles (EVs) that are released by cells play an important role in intercellular communication and are naturally designed for genetic exchange between cells. Here, we show that the EVs have a large beneficial effect in polyethyleneimine (PEI)-mediated transfection of a GFP-encoding plasmid into HEK293T cells. An improvement of transfection efficiency of ~500% and a decrease in toxicity were observed in a specific concentration range of PEI. The EVs also greatly improved the transfection of the same plasmid into zebrafish embryos. To verify the generality of this gene transfection approach, we also tested the cell viability and gene transfection efficiency using two other plasmids (EpTEN and ELuc) and in another cell line (A549). The measured increase in transfection efficiency makes EV a promising candidate for enhancement of the quality of current PEI-based transfection technique.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crys-tals known as hemozoin. The mechanisms underlying this biomi-neralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and in-ternalize a lipocalin-like protein, PV5, to control heme crystalliza-tion. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaf-fected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antima-larial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and under-score the heme biomineralization pathway as an attractive target for therapeutic exploitation.

Modern direct electron detectors (DEDs) provided a giant leap in the use of cryogenic electron microscopy (cryoEM) to study the structures of macromolecules and complexes thereof. However, the currently available commercial DEDs, all based on the monolithic active pixel sensor, still require relative long exposure times and their best results have only been obtained at 300 keV. There is a need for pixelated electron counting detectors that can be operated at a broader range of energies, at higher throughput and higher dynamic range. Hybrid Pixel Detectors (HPDs) of the Medipix family were reported to be unsuitable for cryo-EM at energies above 80 keV as those electrons would affect too many pixels. Here we show that the Timepix3, part of the Medipix family, can be used for cryo-EM applications at higher energies. We tested Timepix3 detectors on a 200 keV FEI Tecnai Arctica microscope and a 300 keV FEI Tecnai G2 Polara microscope. A correction method was developed to correct for per-pixel differences in output. Timepix3 data were simulated for individual electron events using the package Geant4Medipix. Global statistical characteristics of the simulated detector response were in good agreement with experimental results. A convolutional neural network (CNN) was trained using the simulated data to predict the incident position of the electron within a pixel cluster. After training, the CNN predicted, on average, 0.50 pixel and 0.68 pixel from the incident electron position for 200 keV and 300 keV electrons respectively. The CNN improved the MTF of experimental data at half Nyquist from 0.39 to 0.70 at 200 keV, and from 0.06 to 0.65 at 300 keV respectively. We illustrate that the useful dose-lifetime of a protein can be measured within a 1 second exposure using Timepix3.

Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently discovered lugdunomycin (1), a highly rearranged polyketide antibiotic derived from the angucycline backbone that is synthesized via several yet unexplained enzymatic reactions. Here, we show via in vivo, in vitro, and structural analysis that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented C1 ketoreduction. This then sets the stage for the subsequent C-ring cleavage that is key to the rearranged scaffolds of 1. The 1.1 Å structures of LugOII in complex with either ligand 8-O-Methylrabelomycin (4) or 8-O-Methyltetrangomycin (5) and of apoenzyme were resolved, which revealed a canonical Rossman fold and a remarkable conformational change during substrate capture and release. Mutational analysis uncovered key residues for substrate access, position, and catalysis as well as specific determinants that control its dual functionality. The insights obtained in this work hold promise for the discovery and engineering of other promiscuous reductases that may be harnessed for the generation of novel biocatalysts for chemoenzymatic applications.

Compared with X-rays, electron diffraction faces a crucial challenge: dynamical electron scattering compromises structure solution and its effects can only be modelled in specific cases. Dynamical scattering can be reduced experimentally by decreasing crystal size but not without a penalty, as it also reduces the overall diffracted intensity. In this article it is shown that nanometre-sized crystals from organic pharmaceuticals allow positional refinement of the hydrogen atoms, even whilst ignoring the effects of dynamical scattering during refinement. To boost the very weak diffraction data, a highly sensitive hybrid pixel detector was employed. A general likelihood-based computational approach was also introduced for further reducing the adverse effects of dynamic scattering, which significantly improved model accuracy, even for protein crystal data at substantially lower resolution.

Multi-slice simulations of electron diffraction by three-dimensional protein crystals have indicated that structure solution would be severely impeded by dynamical diffraction, especially when crystals are more than a few unit cells thick. In practice, however, dynamical diffraction turned out to be less of a problem than anticipated on the basis of these simulations. Here it is shown that two scattering phenomena, which are usually omitted from multi-slice simulations, reduce the dynamical effect: solvent scattering reduces the phase differences within the exit beam and inelastic scattering followed by elastic scattering results in diffusion of dynamical scattering out of Bragg peaks. Thus, these independent phenomena provide potential reasons for the apparent discrepancy between theory and practice in protein electron crystallography.

Diffraction data have a higher contrast, are less susceptible to sample movement and are electron-optically more robust. Diffraction patterns are insensitive to the sample movement and therefore allow obtaining highest possible resolution. We will discuss the possibilities of electron diffraction data of single protein complexes. The contrast of diffraction patterns can be enhanced by averaging data of similarly oriented molecules, which for welldefined molecular complexes could yield three-dimensional distribution of the scattering potentials with enough resolution for atomic interpretation. In diffraction mode, the phase of the scattered wave is not acquired by the detector and need to be retrieved for the sample structure retrieval. There are several strategies that would allow phasing data collected in diffraction mode and we discuss progress in this area.

Bacteria swim and swarm by rotating the micrometers long, helical filaments of their flagella. They change direction by reversing their flagellar rotation, which switches the handedness of the filament's supercoil. So far, all studied functional filaments are composed of a mixture of L- and R-state flagellin monomers. Here we show in a study of the wild type Firmicute Kurthia sp., that curved, functional filaments can adopt a conformation in vivo that is closely related to a uniform, all-L-state. This sheds additional light on transitions of the flagellar supercoil and uniquely reveals the atomic structure of a wild-type flagellar filament in vivo, including six residues showing clearly densities of O-linked glycosylation.

A Boersch phase plate facilitates in-situ control of electron phase shift by altering the phase of electrons proportionally to the applied electrical potential. A device comprising of multiple such phase shifter elements will be able to modulate the wavefront of a coherent electron beam and control electron interference. In this work, we develop a fabrication method for multi-element electron phase shifter by utilizing state-of-the-art electron beam lithography and reactive ion etching techniques. We fabricated a device with three phase shifter elements in metal-insulator-metal structure and tested its electron transmission characteristics in a transmission electron microscope at beam energy of 200 keV. The experiment confirmed the voltage-controlled evolution of electron interference for individual phase shifter element. We demonstrate the voltage-controlled phase shifting properties of phase shifter elements and analyze the experimental results in comparison with 3-dimensional electrostatic simulation.

Electron diffraction is a relatively novel alternative to X-ray crystallography for the structure determination of macromolecules from three-dimensional nanometre-sized crystals. The continuous-rotation method of data collection has been adapted for the electron microscope. However, there are important differences in geometry that must be considered for successful data integration. The wavelength of electrons in a TEM is typically around 40 times shorter than that of X-rays, implying a nearly flat Ewald sphere, and consequently low diffraction angles and a high effective sample-to-detector distance. Nevertheless, the DIALS software package can, with specific adaptations, successfully process continuous-rotation electron diffraction data. Pathologies encountered specifically in electron diffraction make data integration more challenging. Errors can arise from instrumentation, such as beam drift or distorted diffraction patterns from lens imperfections. The diffraction geometry brings additional challenges such as strong correlation between lattice parameters and detector distance. These issues are compounded if calibration is incomplete, leading to uncertainty in experimental geometry, such as the effective detector distance and the rotation rate or direction. Dynamic scattering, absorption, radiation damage and incomplete wedges of data are additional factors that complicate data processing. Here, recent features of DIALS as adapted to electron diffraction processing are shown, including diagnostics for problematic diffraction geometry refinement, refinement of a smoothly varying beam model and corrections for distorted diffraction images. These novel features, combined with the existing tools in DIALS , make data integration and refinement feasible for electron crystallography, even in difficult cases.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.