Trypanosoma brucei is the causal agent of African trypanosomiasis in humans and animals, the latter resulting in significant negative economic impacts in afflicted areas of the world. Resistance has arisen to the trypanocidal drugs pentamidine and melarsoprol through mutations in the aquaglyceroporin TbAQP2 that prevent their uptake. Here, we use cryogenic electron microscopy to determine the structure of TbAQP2 from T. brucei, bound to either the substrate glycerol or to the sleeping sickness drugs, pentamidine or melarsoprol. The drugs bind within the AQP2 channel at a site completely overlapping that of glycerol. Mutations leading to a drug-resistant phenotype were found in the channel lining. Molecular dynamics (MD) simulations showed the channel can be traversed by pentamidine, with a low energy binding site at the centre of the channel, flanked by regions of high energy association at the extracellular and intracellular ends. Drug-resistant TbAQP2 mutants are still predicted to bind pentamidine, but the much weaker binding in the centre of the channel observed in the MD simulations would be insufficient to compensate for the high energy processes of ingress and egress, hence impairing transport at pharmacologically relevant concentrations. The structures of drug-bound TbAQP2 represent a novel paradigm for drug–transporter interactions and are a new mechanism for targeting drugs in pathogens and human cells.

Cell-cell communication underlies all emergent properties of the brain, including cognition, learning and memory. The physical basis for these communications is the synapse, a multi-component structure requiring coordinated interactions between diverse cell types. However, many aspects of three-dimensional (3D) synaptic organization remain poorly understood. Here, we developed an approach, seamless in situ trimming and milling (SISTM), to reliably fabricate sufficiently thin lamellae for mapping of the 3D nanoarchitecture of synapses in mouse, monkey and human brain tissue under near-native conditions via cryo-electron tomography (cryo-ET). We validated SISTM in a mouse model of Huntington's disease, demonstrating distinct 3D alterations to synaptic vesicles and mitochondria. By successfully applying SISTM to macaque brain, we described the 3D architecture of a tripartite synapse within the cortex. Subtomogram averaging (STA) enabled spatial mapping of astrocyte-neuron contacts within the tripartite synapse, revealing neurexin-neuroligin complexes as potential constituents that tether the two cell types. Finally, we showed that the defining features of synaptic nanoarchitecture were conserved across species and evident in human brain tissue obtained postmortem. Combining SISTM with cryo-ET and STA is a starting point for a new understanding of brain organization, disease-induced structural alterations and the development of rational, structure-guided therapeutics.

LAT1 (SLC7A5) transports large neutral amino acids and plays pivotal roles in cancer proliferation, immune response and drug delivery. Despite recent advances in structural understanding of LAT1, how it discriminates substrates and inhibitors including the clinically relevant drugs remains elusive. Here we report six structures of LAT1 across three conformations with bound ligands, elucidating its substrate transport and inhibitory mechanisms. JPH203 (also known as nanvuranlat or KYT-0353), an anticancer drug in clinical trials, traps LAT1 in an outward-facing state with a U-shaped conformer, with its amino-phenylbenzoxazol moiety pushing against transmembrane helix 3 (TM3) and bending TM10. Physiological substrates like ʟ-Phe lack such effects, whereas melphalan poses steric hindrance, explaining its inhibitory activity. The "classical" system L inhibitor BCH induces an occluded state critical for transport, confirming its substrate-like behavior. These findings provide a structural basis for substrate recognition and inhibition of LAT1, guiding future drug design.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Electron tomography of frozen, hydrated samples allows structure determination of macromolecular complexes that are embedded in complex environments. Provided that the target complexes may be localised in noisy, three-dimensional tomographic reconstructions, averaging images of multiple instances of these molecules can lead to structures with sufficient resolution for de novo atomic modelling. Although many research groups have contributed image processing tools for these tasks, a lack of standardisation and interoperability represents a barrier for newcomers to the field. Here, we present an image processing pipeline for electron tomography data in RELION-5, with functionality ranging from the import of unprocessed movies to the automated building of atomic models in the final maps. Our explicit definition of metadata items that describe the steps of our pipeline has been designed for interoperability with other software tools and provides a framework for further standardisation.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein/nucleic-acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: E. coli beta-galactosidase with inhibitor, SARS-CoV-2 RNA-dependent RNA polymerase with covalently bound nucleotide analog, and SARS-CoV-2 ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. We found that (1) the quality of submitted ligand models and surrounding atoms varied, as judged by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics, and contact scores, and (2) a composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

An open-source Python library EMDA for cryo-EM map and model manipulation is presented with a specific focus on validation. The use of several functionalities in the library is presented through several examples. The utility of local correlation as a metric for identifying map-model differences and unmodeled regions in maps, and how it is used as a metric of map-model validation is demonstrated. The mapping of local correlation to individual atoms, and its use to draw insights on local signal variations are discussed. EMDA's likelihood-based map overlay is demonstrated by carrying out a superposition of two domains in two related structures. The overlay is carried out first to bring both maps into the same coordinate frame and then to estimate the relative movement of domains. Finally, the map magnification refinement in EMDA is presented with an example to highlight the importance of adjusting the map magnification in structural comparison studies.

The number of high-resolution structure determination by cryo-EM single particle analysis (SPA) is growing rapidly.Focusing on maps having better resolution than 3 Å deposited in the Electron Microscopy Data Bank (EMDB), there were 316 depositions in 2019 while it was 82 in 2018.It increases the importance of method developments for accurate determination of atomic coordinates and thus the model validation, where not only the geometric quality but also the fitness to the map is of great importance.Here we present a new program Servalcat for the refinement and map calculation of cryo-EM SPA structures.Servalcat implements a refinement pipeline using REFMAC5, which uses a dedicated likelihood function for SPA [1].It takes as inputs unsharpened and unweighted half maps from independent reconstructions.The variance of noise in Fourier coefficients is estimated using the half maps.A weighted and sharpened Fo-Fc map is calculated after the refinement.The Fourier coefficients for the difference maps are derived as expectation values of unknown Fourier coefficients using their posterior distribution given observations and model parameters.Refinement of atomic displacement parameters is crucial for calculation of a sensible Fo-Fc map.It was shown to be useful for visualization of weak features like hydrogen atoms and model errors as it is done routinely in crystallography.Although hydrogen densities are weaker than heavier atoms (e.g.C, N, O), they are stronger than in the electron density maps produced by Xray crystallography, and some hydrogen atoms are even visible at ~1.8 Å.About half of the EMDB-deposited SPA maps have non-C1 point group symmetry.If the map has been symmetrised during reconstruction, then all downstream programs should be aware of it and the atomic structure model must follow the symmetry.A user can give an asymmetric unit model and a point group symbol to Servalcat for refinement.The NCS constraint function in REFMAC5 was updated to consider non-bonded interactions and ADP similarity restraints between symmetry copies.The MTRIX records in the PDB format and _struct_ncs_oper in the mmCIF format are used to encode the symmetry information.Currently, there are only few asymmetric unit model depositions to the PDB except viruses.We think that refining and depositing asymmetric unit models with annotations of symmetry will be a common practice in future.We are also developing a new program EMDA, for cryo-EM map and model manipulation with the main focus on validation.EMDA offers several metrics for map validation including FSC combined with mask correction by high resolution noise substitution [2], local correlation using half maps, optimal alignment between maps and magnification scaling using maximum-likelihood method.Also, EMDA includes metrics for map-model validation such as local correlation between map-and-model, which can be used to investigate the quality of the map-to-model fit.Both EMDA [3] and Servalcat [4] are freely available under an open-source licence.They are also available within the CCP-EM package.

Cryo-EM is becoming an increasingly popular method of structure determination in structural biology.As the number of cryo-EM structures increases, it is important to maintain standards that measure the quality of those structures.The correctness of atomic models is very important because they often serve as targets for novel drugs or the knowledge base of such developments.Also, such standards are important to prevent the accumulation of errors of the structures in the databases.Thus, careful curation and validation of cryo-EM maps and derived atomic models are of utmost importance.We have developed the EMDA Python package [1] that includes tools for cryo-EM map and model manipulation.In this presentation, the emphasis is given to those for validation.The majority of the current validation tools used in single-particle cryo-EM analyses are global metrics.They provide summaries of the global quality of maps or map-model fits.In order to reveal the local variation of the signal in maps and map-model fits, a new set of tools based on the local correlation have been developed.To calculate the local correlation, a spherical kernel is convolved with the map in image space to yield a correlation value at each voxel resulting in a three-dimensional (3D) correlation map.The variation of calculated correlation depends on the size of the kernel.The local correlation calculated using half maps captures the local variations in the signal, whereas the local correlation calculated between a map and a model indicates the quality of their fit.Mapmodel local correlation can be used to identify model regions outside the density or poorly fitted.Also, it can highlight unmodeled regions on the map.While the half map local correlation is useful to identify the presence/absence of the signal, its comparison with the map-model local correlation can be used to validate the map-model fit.In this presentation, we will demonstrate the use of local correlation through several examples.EMDA includes several tools based on the maximum likelihood method.EMDA's map-overlay and map magnification refinement are based on maximisation of the joint probability distribution between two maps by a quasi-Newton method.We will demonstrate the use of map overlay and magnification refinement implemented in EMDA through examples.

At the Swiss Light Source macromolecular crystallography (MX) beamlines the collection of serial synchrotron crystallography (SSX) diffraction data is facilitated by the recent DA+ data acquisition and analysis software developments. The SSX suite allows easy, efficient and high-throughput measurements on a large number of crystals. The fast continuous diffraction-based two-dimensional grid scan method allows initial location of microcrystals. The CY+ GUI utility enables efficient assessment of a grid scan's analysis output and subsequent collection of multiple wedges of data (so-called minisets) from automatically selected positions in a serial and automated way. The automated data processing ( adp ) routines adapted to the SSX data collection mode provide near real time analysis for data in both CBF and HDF5 formats. The automatic data merging ( adm ) is the latest extension of the DA+ data analysis software routines. It utilizes the sxdm (SSX data merging) package, which provides automatic online scaling and merging of minisets and allows identification of a minisets subset resulting in the best quality of the final merged data. The results of both adp and adm are sent to the MX MongoDB database and displayed in the web-based tracker, which provides the user with on-the-fly feedback about the experiment.

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.

De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Here we present a method that combines in situ serial crystallography with de novo phasing for fast, efficient membrane protein structure determination. The method enables systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without the need for direct crystal harvesting. The requisite data quality for experimental phasing is achieved by accumulating diffraction signals from isomorphous crystals identified post-data collection. The method works in all experimental phasing scenarios and is particularly attractive with fragile, weakly diffracting microcrystals. The automated serial data collection approach can be readily adopted at most microfocus macromolecular crystallography beamlines.

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 x 75 mu m, frame rates of up to 3000 Hz and a dead time as low as 3.8 mu s. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine phi-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed.

Pentaindenocorannulene (C50 H20  , 1), a deep bowl polynuclear aromatic hydrocarbon, accepts 4 electrons, crystallizes in columnar bowl-in-bowl assemblies and forms a nested C60 @12 complex. Spectra, structures and computations are presented.

The ability to avoid radiation damage with femtosecond X-ray pulses is a powerful argument for the use of X-ray free electron lasers (XFELs), with the important caveat that the observed diffraction consists entirely of partially recorded Bragg spots.Correcting the structure factor intensity to the full spot equivalent improves the quality of the final, merged data set that is created by mutually scaling hundreds or thousands of separate shots.However, several years after the initial XFEL results, efforts are still ongoing to produce optimal models of the spot partiality, with programs such as cctbx.xfel,DIALS, Prime, and ccpxfel each taking a slightly different approach.Data merging affects the final outcome, including the ability to phase with anomalous scattering data, and to discern high-resolution structural details.Examples will be shown from recent data collection at LCLS, including results from the new protein crystallography end station MFX.

The β-phase of the industrially important Pigment Red 170 (β-P.R. 170) has a structure with severe layer stacking disorder. The single-crystal X-ray diffraction pattern consists of a difficult-to-disentangle mix of Bragg diffraction superimposed on rods of diffuse scattering which impede the estimation of accurate Bragg intensities. Two average monoclinic structure models with the same unit-cell dimensions, but different extents of disorder in the layers and different space groups seem plausible, one with the non-conventional space group setting B2(1)/g (No. 14, Z' = 2) and one in P2(1)/a (No. 14, Z' = 4). Disordered molecules related by a translation of 0.158b are present in all layers of the B2(1)/g model and in every second layer of the P2(1)/a model. Layer-to-layer contacts are practically the same in both models. According to order-disorder theory, both models are valid superposition structures. Structure-factor calculations show that the pattern of strong and weak Bragg reflections is very similar for the two models. R factors indicate that the B2(1)/g model is the most economic representation of the average structure. However, given the limitations in data processing, the P2(1)/a model should not be discarded and further insight sought from a detailed analysis of the experimental diffuse scattering. The difficulties encountered in this analysis raise the question of whether or not the concept of an average structure is applicable in practice to β-P.R. 170.