Phillip James Stansfeld

The Protein Data Bank in Europe (PDBe; pdbe.org) provides open access to experimentally determined biomacromolecular structures and enriches these data with biological and functional context. Here, we describe recent developments that expand the information connected to PDB structures and introduce new ways for researchers and computational tools to access it. Literature integration has been extended to additional open-access publications, substantially increasing the availability of residue-level annotations mined from the scientific literature. Cross-links to molecular dynamics simulations in MDposit have been established, and simulation-derived residue-level annotations, including relative solvent accessibility and membrane-related properties, have been integrated into PDBe-KB (pdbekb.org). PDBe entry pages now expose additional experimental and sequence information, links to related resources, and extended PDB identifiers. Protein channel, tunnel, and pore analyses generated using MOLE have also been incorporated into our weekly release pipeline and made available for download. Improved API documentation facilitates the discovery of data fields and search parameters, while three Model Context Protocol servers facilitate agentic access to the PDBe APIs, Search, and the PDBe-KB knowledge graph. Together, these developments make structural data and its wider biological context easier to discover, interpret, and reuse through both conventional and agentic workflows.

Membrane proteins engage in dynamic transient interactions with surrounding lipids along with their substrates. Capturing these concomitant contacts often remains a challenging problem. Here, we demonstrate that generative deep learning-designed WRAP domains can preserve weak interactions of membrane proteins in native mass spectrometry. Using WRAP-fused GlpG, AqpM, and OmpA as model systems, we show that lipids can bind in multiple positions inside the WRAP, allowing for their characterization by native mass spectrometry. We show that lipid interactions can be altered by changing the WRAP structure. We use the approach to show that WRAP–OmpA binds phosphatidylethanolamine lipids within cavities formed at the protein–WRAP interface, while simultaneously accommodating weak chitobiose ligand binding. These findings establish WRAPs as versatile vehicles to probe ligand and lipid interactions of membrane proteins.

The outer membrane of Gram-negative bacteria is assembled and maintained by the lipopolysaccharide (LPS) transport machinery, which extracts LPS from the inner membrane and conveys it across the periplasm via a protein bridge. How the inner-membrane transporter, LptB ₂ FGC, engages the soluble bridge component, LptA (named LptH in Pseudomonas aeruginosa ), and how this junction responds to the ATPase cycle remain unresolved. Here, we determine cryo-electron microscopy structures of the P. aeruginosa LptB ₂ FGC-LptH complex in nucleotide-free and AMP-PNP-bound states at 3.24 Å and 3.05 Å resolution, respectively. The structures show that LptH augments the terminal beta-strand of LptC, thereby completing a continuous beta-jellyroll conduit. ATP analogue binding closes the LptB dimer and the transmembrane LPS-binding cavity while remodelling the LptF-LptC junction at the base of the assembled bridge. Molecular dynamics simulations show that the LptC-LptH connection remains intact but becomes less compact in the ATP-bound ensemble. Native mass spectrometry further reveals that thanatin preferentially disrupts LptH-LptH and LptC-LptH assemblies. Together, these findings establish the architecture of the transporter-bridge junction and reveal how nucleotide-driven closure remodels an already assembled periplasmic conduit while preserving bridge continuity.

Molecular organization of the plasma membrane at nano- and micrometer scales is critical for its function in all living cells. This emerges not only from the self-assembly of lipids and proteins but also from active forces originating in the underlying cytoskeletal cortex. These forces drive membrane molecules into nonequilibrium steady-state patterns such as nanoclusters. However, the molecular agents connecting membrane organization with cytoskeletal dynamics and stresses have remained unknown. Here, we show that two classes of ubiquitous ancestral nonmuscle myosins are deployed for the organization of different types of membrane components. Inner leaflet–localized class I myosins link outerleaflet glycosylphosphatidylinositol-anchored molecules to juxta-membrane actin filaments, whereas the more cortically localized Class II myosins operate on transmembrane proteins endowed with actin-binding capacity. Consistent with an active Flory-Huggins theory for phase separation, these observations show that the distinct motor-driven membrane molecules generate spatially segregated mesoscale domains, enriched in nanoclusters derived from different myosin classes. Moreover, chemically reversible posttranslational modifications such as palmitoylation enable concatenation of these domains by enhancing the affinity of the membrane domain constituents for each other. We anticipate that the segregation potential of the adenosine 5′-triphosphate (ATP)–fueled cell membrane is made available for the crucial purpose of modulating information transduction because it can be regulated in space and time during the construction of signaling cascades, underpinning functional plasma membrane organization.

Membrane proteins engage in dynamic transient interactions with surrounding lipids along with their substrates. Capturing these concomitant contacts often remains a challenging problem. Here, we demonstrate that generative deep learning-designed WRAP domains can preserve weak interactions of membrane protein complexes in native mass spectrometry. Using WRAP-fused GlpG, AqpM, and OmpA as model systems, we show that lipid interactions can be retained and characterized without detergent micelles. We use the approach to show that WRAP-OmpA selectively binds phosphatidylethanolamine lipids within cavities formed at the protein–WRAP interface, while simultaneously accommodating weak chitobiose ligand binding. These findings establish WRAPs as versatile vehicles to probe ligand and lipid interactions of membrane proteins.

The twin-arginine translocation (Tat) system is a mechanistically unique protein transport pathway moving folded proteins across membranes. It is found in all domains of life and is essential for bacterial virulence and plant photosynthesis. The membrane proteins, TatA, TatB and TatC form a core complex to which substrate proteins bind, triggering the recruitment of additional TatA protomers to form the transport site. Here we present cryo-electron microscopy structures of the prototypical TatBC complex from Escherichia coli and the atypical complexes from Nitratifactor salsuginis and Myxococcus xanthus in a resting state, alongside TatAC substrate-bound TatBC and TatABC complexes from E. coli in the early stages of transport. These structures demonstrate that substrate proteins associate with the core complex solely through their N-terminal signal peptides. The Tat targeting sequences of the signal peptides make specific contacts with TatC, and the peptide body is clamped by TatB. The core complex contains highly tilted transmembrane helices that drive extreme local membrane thinning. On the basis of our structures and biochemical and functional analyses, we propose a model for the early steps in Tat transport.

The outer membrane is vital for Gram-negative bacteria, playing crucial roles in colonization, pathogenesis and drug resistance. The translocation and assembly module A and B (TamAB) nanomachinery has been reported to be involved in transport of phospholipids from the inner membrane to the outer membrane, as well as insertion of critical outer membrane proteins. However, the underlying mechanisms remain poorly understood. Here we report cryogenic electron microscopy structures of TamAB in two conformations at resolutions of 3.69 and 3.82 Å. We reveal a hybrid barrel structure formed between the first β-strand of the TamA barrel and the last β-strand of the TamB C-terminal domain, which is folded inside the β-barrel. By integrating structural analysis with functional data, biochemical assays, and molecular dynamics simulations, we identify key residues involved in TamAB interactions and characterize the mechanisms of anterograde phospholipid transport within the continuously beta-helical hydrophobic cavity of TamB. Through disulfide bond crosslinking and functional assays, we reveal that TamA crosslinks with both TamB and Ag43. Additionally, we confirm that the two cryo-EM conformational states of TamAB exist in vivo. While BAM overexpression can compensate for TamAB deletion in Ag43 insertion, it does not rescue phospholipid transport. Given that TamA and TamB orthologs are widely distributed in among bacterial and eukaryotic organisms, our findings have broad implications in cell envelope biogenesis and offer potential avenues for therapeutic development through inhibition.

Membrane proteins play a vital role in numerous cellular processes, including ion transport, intercellular communication, and antibiotic resistance. Ensuring their accurate orientation within lipid bilayers is essential for reliable molecular simulations and structural analyses. We introduce MemPrO, a robust tool designed to orient a wide range of membrane-associated proteins with precision. While existing tools perform well in standard scenarios, they often struggle with complex configurations such as multibilayer systems or peripheral membrane proteins. MemPrO addresses these limitations by delivering detailed orientation data for proteins in both single and double membrane environments. In addition to mapping spatial arrangements relative to lipid bilayers, MemPrO incorporates advanced analytical features, including membrane curvature analysis. It also extends its capability to predict the position and orientation of the bacterial cell wall. By integrating these features, MemPrO significantly enhances our ability to model and interpret the intricate architecture of cellular envelopes. This supports both fundamental research and the development of novel therapeutic strategies through a deeper understanding of membrane-protein interactions.

Gram-negative bacteria are intrinsically resistant to many antibiotics because of densely packed lipopolysaccharides (LPS) in the outer leaflet of their outer membrane (OM), which acts as a highly effective barrier towards the spontaneous permeation of toxic molecules, including antibiotics. LPS are extracted from the inner membrane by the ABC transporter LptB ₂ FGC and translocated across the periplasm via a protein bridge to the OM. While structural studies have elucidated aspects of Lpt function in enterobacteria, little is known about how this system operates in divergent species such as Pseudomonas aeruginosa , a major human pathogen. Here, we report five cryo-electron microscopy structures of P. aeruginosa LptB ₂ FG and LptB ₂ FGC, revealing a rigid body movement in the periplasmic β-jellyroll domains necessary for LPS to shuttle through the periplasmic space. Notably, these structures exhibit a significantly smaller LPS binding cavity compared to previously determined models, suggesting the ligand-unbound states of the transporter. Mass spectrometry and molecular dynamics simulations indicate that the phosphate groups of LPS are the key determinants for binding and that the transporter can also accommodate cardiolipin. Together, these findings reveal previously unappreciated structural diversity in the Lpt system and provide mechanistic insight into how pathogenic Gram-negative bacteria tailor LPS recognition and transport. This understanding offers new avenues for the development of novel inhibitors targeting membrane biogenesis.

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.