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IHMCIF: an extension of the PDBx/mmCIF data standard for integrative structure determination methods
(2024)
IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.
The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic field’s methods to determine a robotic arm’s forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.
Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of ATP. Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt-bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.
Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of ATP. Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt-bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. Here, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100-fold above the noise level. We establish a TM pipeline with systematically tuned parameters for automated, objective and comprehensive feature identification. High-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, lipid membranes and microtubules, and individual subunits, demonstrate that TM is generic. We resolve ~100-kDa proteins, connect the functional states of complexes to their cellular localization, and capture vaults carrying ribosomal cargo in situ. By capturing individual molecular events inside living cells with defined statistical confidence, high-confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.
Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments
(2023)
The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying Pi release from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a ‘molecular backdoor’. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and only opens transiently through concerted backbone movements and rotameric rearrangements of residues close to the nucleotide binding pocket. This mechanism explains why Pi escapes rapidly from the filament end and yet slowly from internal actin subunits. In an actin variant associated with nemaline myopathy, the backdoor is predominantly open in filament-core subunits, resulting in greatly accelerated Pi release after polymerization and filaments with drastically shortened ADP-Pi caps. This demonstrates that the Pi release rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between Pi and Mg2+ at the nucleotide-binding site. Our results provide the molecular basis for Pi release from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament.
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are major facilitator superfamily transporters from the solute carrier family 49. Dysregulation of these ubiquitous transporters has been linked to various haematological and neurological disorders. While both FLVCRs were initially proposed to hold a physiological function in heme transport, subsequent studies questioned this notion. Here, we used structural, computational and biochemical methods and conclude that these two FLVCRs function as human choline transporters. We present cryo-electron microscopy structures of FLVCRs in different inward- and outward-facing conformations, captured in the apo state or in complex with choline in their translocation pathways. Our findings provide insights into the molecular framework of choline coordination and transport, largely mediated by conserved cation-π interactions, and further illuminate the conformational dynamics of the transport cycle. Moreover, we identified a heme binding site on the protein surface of the FLVCR2 N-domain, and observed that heme actively drives the conformational transitions of the protein. This auxiliary binding site might indicate a potential regulatory role of heme in the FLVCR2 transport mechanisms. Our work resolves the contested substrate specificity of the FLVCRs, and sheds light on the process of maintaining cellular choline homeostasis at the molecular level.
More than 75% of surface and secreted proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, influencing protein dynamics and interactions with other molecules. Glycan conformational freedom impairs complete structural elucidation of glycoproteins. Computer simulations may be used to model glycan structure and dynamics. However, such simulations typically require thousands of computing hours on specialized supercomputers, thus limiting routine use. Here, we describe a reductionist method that can be implemented on personal computers to graft ensembles of realistic glycan conformers onto static protein structures in a matter of minutes. Using this open-source pipeline, we reconstructed the full glycan cover of SARS-CoV-2 Spike protein (S-protein) and a human GABAA receptor. Focusing on S-protein, we show that GlycoSHIELD recapitulates key features of extended simulations of the glycosylated protein, including epitope masking, and provides new mechanistic insights on N-glycan impact on protein structural dynamics.
TriMem: A parallelized hybrid Monte Carlo software for efficient simulations of lipid membranes
(2022)
Lipid membranes are integral building blocks of living cells and perform a multitude of biological functions. Currently, molecular simulations of cellular-scale membrane remodeling processes at atomic resolution are extremely difficult, due to their size, complexity, and the large times-scales on which these processes occur. Instead, elastic membrane models are used to simulate membrane shapes and transitions between them and to infer their properties and functions. Unfortunately, an efficiently parallelized open-source simulation code to do so has been lacking. Here, we present TriMem, a parallel hybrid Monte Carlo simulation engine for triangulated lipid membranes. The kernels are efficiently coded in C++ and wrapped with Python for ease-of-use. The parallel implementation of the energy and gradient calculations and of Monte Carlo flip moves of edges in the triangulated membrane enable us to simulate large and highly curved membrane structures. For validation, we reproduce phase diagrams of vesicles with varying surface-to-volume ratios and area difference. We also compute the density of states to verify correct Boltzmann sampling. The software can be used to tackle a range of large-scale membrane remodeling processes as a step toward cell-scale simulations. Additionally, extensive documentation make the software accessible to the broad biophysics and computational cell biology communities.