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.

DNA gyrase is a bacterial type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology and an archetypical target of antibiotics. The widely used quinolone class of drugs use a water-metal ion bridge in interacting with the GyrA subunit of DNA gyrase. Zoliflodacin sits in the same pocket as quinolones but interacts with the GyrB subunit and also stabilizes lethal double-stranded DNA breaks. Gepotidacin has been observed to sit on the twofold axis of the complex, midway between the two four-base-pair separated DNA-cleavage sites and has been observed to stabilize singe-stranded DNA breaks. Here, we use information from three crystal structures of complexes of Staphlococcus aureus DNA gyrase (one with a precursor of gepotidacin and one with the progenitor of zoliflodacin) to propose a simple single moving metal-ion-catalyzed DNA-cleavage mechanism. Our model explains why the catalytic tyrosine is in the tyrosinate (negatively charged) form for DNA cleavage. Movement of a single catalytic metal-ion (Mg2+ or Mn2+) guides water-mediated protonation and cleavage of the scissile phosphate, which is then accepted by the catalytic tyrosinate. Type IIA topoisomerases need to be able to rapidly cut the DNA when it becomes positively supercoiled (in front of replication forks and transcription bubbles) and we propose that the original purpose of the small Greek Key domain, common to all type IIA topoisomerases, was to allow access of the catalytic metal to the DNA-cleavage site. Although the proposed mechanism is consistent with published data, it is not proven and other mechanisms have been proposed. Finally, how such mechanisms can be experimentally distinguished is considered.

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.

Proteins frequently undergo covalent modification at the post-translational level, which involves the covalent attachment of chemical groups onto amino acids. This can entail the singular or multiple addition of small groups, such as phosphorylation; long-chain modifications, such as glycosylation; small proteins, such as ubiquitination; as well as the interconversion of chemical groups, such as the formation of pyroglutamic acid. These post-translational modifications (PTMs) are essential for the normal functioning of cells, as they can alter the physicochemical properties of amino acids and therefore influence enzymatic activity, protein localization, protein–protein interactions and protein stability. Despite their inherent importance, accurately depicting PTMs in experimental studies of protein structures often poses a challenge. This review highlights the role of PTMs in protein structures, as well as the prevalence of PTMs in the Protein Data Bank, directing the reader to accurately built examples suitable for use as a modelling reference.

One of the challenges for experimental structural biology in the 21st century is to see chemical reactions happen. Staphylococcus aureus (S. aureus) DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology. Drugs, such as gepotidacin, zoliflodacin and the quinolone moxifloxacin, can stabilize these normally transient DNA strand breaks and kill bacteria. Crystal structures of uncleaved DNA with a gepotidacin precursor (2.1 Å GSK2999423) or with doubly cleaved DNA and zoliflodacin (or with its progenitor QPT-1) have been solved in the same P61 space-group (a = b ≈ 93 Å, c ≈ 412 Å). This suggests that it may be possible to observe the two DNA cleavage steps (and two DNA-religation steps) in this P61 space-group. Here, a 2.58 Å anomalous manganese dataset in this crystal form is solved, and four previous crystal structures (1.98 Å, 2.1 Å, 2.5 Å and 2.65 Å) in this crystal form are re-refined to clarify crystal contacts. The structures clearly suggest a single moving metal mechanism—presented in an accompanying (second) paper. A previously published 2.98 Å structure of a yeast topoisomerase II, which has static disorder around a crystallographic twofold axis, was published as containing two metals at one active site. Re-refined coordinates of this 2.98 Å yeast structure are consistent with other type IIA topoisomerase structures in only having one metal ion at each of the two different active sites.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance Statement Oceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

Metals are essential components for the structure and function of many proteins. However, accurate modelling of their coordination environments remains a challenge due to the complexity and diversity of metal-coordination geometries. To address this, a method is presented for extracting and analysing coordination information, including bond lengths and angles, from the Crystallography Open Database. By using these data, comprehensive descriptions of metal-containing components are generated. A stereochemical information generator for a particular component within a specific macromolecule leverages an example PDB/mmCIF file containing the component to account for the actual surrounding environment. A matching process has been developed and implemented to align the derived metal structures with idealized coordinates from a coordination geometry library. Additionally, various strategies, depending on the quality of the matches, were employed to compile distance and angle statistics for the refinement of macromolecular structures. The developed methods were implemented in a new program, MetalCoord, that classifies and utilizes the metal-coordination geometry. The effectiveness of the developed algorithms was tested using metal-containing components from the PDB. As a result, metal-containing components from the CCP4 monomer library have been updated. The updated monomer dictionaries, in concert with the derived restraints, can be used in most structural biology computations, including macromolecular crystallography, single-particle cryo-EM and even molecular mechanics.

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.

Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand-recognition processes and protein–protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localization of only the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with REFMAC5 afforded models characterized by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium–low resolution.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research on this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the D. radiodurans S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjacent hexamer, resulting in a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled D. radiodurans cells, we have obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium D. radiodurans shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting common features in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.

Macromolecular refinement uses experimental data together with prior chemical knowledge (usually digested into geometrical restraints) to optimally fit an atomic structural model into experimental data, while ensuring that the model is chemically plausible. In the CCP4 suite this chemical knowledge is stored in a Monomer Library, which comprises a set of restraint dictionaries. To use restraints in refinement, the model is analysed and template restraints from the dictionary are used to infer (i) restraints between concrete atoms and (ii) the positions of riding hydrogen atoms. Recently, this mundane process has been overhauled. This was also an opportunity to enhance the Monomer Library with new features, resulting in a small improvement in REFMAC5 refinement. Importantly, the overhaul of this part of CCP4 has increased flexibility and eased experimentation, opening up new possibilities.

AbstractHydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand recognition processes, and protein-protein interactions. However, their direct visualisation by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localisation of the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher), that is not often attainable. Differently, neutron diffraction methods enable the visualisation of most H atoms, typically in the form of deuterium (D) atoms at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought.REFMAC5 of the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of theCCP4 Monomer Library, the source of prior chemical information used in refinement. One new feature for neutron data analysis inREFMAC5 is the refinement of the protium/deuterium (1H/D) fraction. This parameter describes the relative1H/D contribution to neutron scattering for H atoms. The newly developedREFMAC5algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) by using either (i) neutron data-only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement withREFMAC5afforded models characterised byR-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy especially for structures at medium-low resolution.SynopsisThe macromolecular refinement packageREFMAC5 of theCCP4 suite has been extended with the incorporation of algorithms for neutron crystallography.

Hydrogen (H) atoms represent a large fraction of the total atomic content of macromolecules.They often play critical roles in enzymatic reactions, as they are involved in the protonation of active site residues, in the organization and orientation of the solvent network, and in the establishment of hydrogen bonds.However, the direct visualisation of H atoms in macromolecules is not straightforward.Since their contribution to the total scattering is minimal owing to their low electron content, only a small percentage of H atoms can be located using X-ray macromolecular crystallography (MX), even at sub-atomic resolution (<1.2 Å).In contrast with MX, neutron macromolecular crystallography (NMX) relies on the interaction between neutrons and atomic nuclei.Ratio of atomic scattering factors of deuterium (D) to that of typical heavy macromolecular atoms such as oxygen, carbon and nitrogen is not far from 1 (1.15, 1.08 and 0.71 respectively).Therefore, the observation of H atoms (typically in the form of D) is possible even at modest resolution (2.0-2.5 Å).Despite the major limitations of this technique, especially due to the low flux of neutron beams, NMX studies are particularly useful in answering research questions that require specific knowledge of H atom positions with the view of obtaining mechanistic insight.Whilst NMX maps show the nuclear positions of H atoms, MX maps indicate the positions of valence-electron density for H atoms shifted along their bond vector.Cryo-electron microscopy (cryo-EM) and electron diffraction experiments inform on both nuclear and electron localisation of H atoms.This project aims at modelling and refining H atoms by using different experimental data (cryo-EM, NMX and electron diffraction) integrated in a common framework, to provide new insights in biological processes such as enzyme mechanisms.This contribution will describe the recent developments in the crystallographic refinement package REFMAC5 [1], for the refinement of structural models obtained by NMX data.The CCP4 Monomer Library [2], typically used as the source for prior chemical information in REFMAC5 and other programs such as Coot [3], has been recently extended to include accurate H atom nucleus to parent atom distances derived from neutron data analysis [4] and quantum mechanics (QM) calculations.New features for the refinement of mixture H/D parameters in NMX and the use of reference structure restraints will be presented.

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.

Recent technological advances in electron cryo-microscopy (cryo-EM) have led to significant improvements in the resolution of many single-particle reconstructions and a sharp increase in the number of entries released in the Electron Microscopy Data Bank (EMDB) every year, which in turn has opened new possibilities for data mining. Here we present a resolution-dependent library of rotamer-specific amino-acid map motifs mined from entries in the EMDB archive with reported resolution between 2.0 and 4.0Å. We further describe 3D-Strudel, a method for map/model validation based on these libraries. 3D-Strudel calculates linear correlation coefficients between the map values of a map-motif from the library and the experimental map values around a target residue. We also present “Strudel Score”, a plug-in for ChimeraX, as a user-friendly tool for visualisation of 3D-Strudel validation results.

The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR’s PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.

In 2020, cryo-EM single particle analysis achieved true atomic resolution, thanks to technological developments in hardware and software. The number of high resolution reconstructions continues to grow, increasing the importance of accurate determination of atomic coordinates. Here, a new Python package and program calledServalcatis presented that is designed to facilitate atomic model refinement.Servalcatimplements a refinement pipeline, using the programREFMAC5 from theCCP4package. After the refinement,Servalcatcalculates a weightedFo−Fcdifference map, which was derived from Bayesian statistics. This map helps manual and automatic model building in real space, as is common practice in crystallography. TheFo−Fcmap helps visualisation of weak features including hydrogen densities. Although hydrogen densities are weak, they are stronger than in electron density maps produced by X-ray crystallography, and some hydrogen atoms are even visible at ∼ 1.8 Å resolution.Servalcatalso facilitates atomic model refinement under symmetry constraints. If a point group symmetry has been applied to the map during reconstruction, the asymmetric unit model is refined with appropriate symmetry constraints.

Covalent linkages between constituent blocks of macromolecules and ligands have been subject to inconsistent treatment during the model-building, refinement and deposition process. This may stem from a number of sources, including difficulties with initially detecting the covalent linkage, identifying the correct chemistry, obtaining an appropriate restraint dictionary and ensuring its correct application. The analysis presented herein assesses the extent of problems involving covalent linkages in the Protein Data Bank (PDB). Not only will this facilitate the remediation of existing models, but also, more importantly, it will inform and thus improve the quality of future linkages. By considering linkages of known type in the CCP4 Monomer Library (CCP4-ML), failure to model a covalent linkage is identified to result in inaccurate (systematically longer) interatomic distances. Scanning the PDB for proximal atom pairs that do not have a corresponding type in the CCP4-ML reveals a large number of commonly occurring types of unannotated potential linkages; in general, these may or may not be covalently linked. Manual consideration of the most commonly occurring cases identifies a number of genuine classes of covalent linkages. The recent expansion of the CCP4-ML is discussed, which has involved the addition of over 16 000 and the replacement of over 11 000 component dictionaries using AceDRG. As part of this effort, the CCP4-ML has also been extended using AceDRG link dictionaries for the aforementioned linkage types identified in this analysis. This will facilitate the identification of such linkage types in future modelling efforts, whilst concurrently easing the process involved in their application. The need for a universal standard for maintaining link records corresponding to covalent linkages, and references to the associated dictionaries used during modelling and refinement, following deposition to the PDB is emphasized. The importance of correctly modelling covalent linkages is demonstrated using a case study, which involves the covalent linkage of an inhibitor to the main protease in various viral species, including SARS-CoV-2. This example demonstrates the importance of properly modelling covalent linkages using a comprehensive restraint dictionary, as opposed to just using a single interatomic distance restraint or failing to model the covalent linkage at all.