Biology subjects

Moss, F. R.

Publications and source records attributed to Moss, F. R..

8 recordsLinked to original sources

bioRxiv2025

Comparing Multislice Simulations of MD Simulations with CryoEM Exposes Membrane Prediction Errors

Cryo-electron microscopy (cryoEM) is a powerful tool for atomic- and molecular-resolution structure determination, while molecular dynamics (MD) simulations are similarly powerful tools for predicting molecular trajectories. Given the challenges in estimating biomolecule dynamics with cryoEM alone, MD simulations are employed to forecast molecular motions and to interpret cryoEM reconstructions. Few methods, however, can evaluate MD predictions directly. Here, we use multislice wave propagation to project sampled snapshots of MD trajectories, either coarse-grained (CG) or all-atom (AA), into simulated cryoEM 3D reconstructions. We compared simulated and experimental images of low- and high-curvature membranes to show that MD simulations qualitatively reflect the fluidity and thus the contrast of biological membranes observed by cryoEM. MD simulations also correctly predicted bilayer dimensions for single component flat bilayers observed in cryoEM images. However, Martini3 CG-MD simulations failed to predict changes in membrane thickness induced by high curvature and with heterogeneous lipid compositions. We pinpointed the misbehavior of polyunsaturated lipid tails and cholesterol in Martini3 simulations as the main error sources contributing to inaccurate bilayer thicknesses. Our comparisons also explain membrane structure discrepancies between cryoEM and small angle X-ray scattering (SAXS). Further testing of MD predictions by direct comparisons between simulated and experimental cryoEM images should lead to the development of more accurate MD force fields. Statement of SignificanceMolecular dynamics (MD) simulations are frequently employed to predict the dynamics of biological macromolecules and assemblies, but these predictions remain difficult to validate experimentally. Cryo-electron microscopy (cryoEM) can be used to directly image the conformational ensemble of macromolecules, but images of Coulombic potential cannot be easily compared to snapshots of atoms from MD simulations. Here, we show that a physics-based multislice image projection algorithm accurately converts MD trajectories to simulated cryoEM 2D images and 3D reconstructions. Using this approach, we identify consistencies and discrepancies between MD simulations and cryoEM experiments. Notably, coarse-grained MD performs poorly compared to all-atom MD when simulating membranes composed of mixtures of lipids that include cholesterol and polyunsaturated lipids, providing observables for MD force field improvement.

biophysics↗

bioRxiv2024

Cardiolipin clustering promotes mitochondrial membrane dynamics

Cardiolipin (CL) is a mitochondria-specific phospholipid that forms heterotypic interactions with membrane-shaping proteins and regulates the dynamic remodeling and function of mitochondria. However, the precise mechanisms through which CL influences mitochondrial morphology are not well understood. In this study, employing molecular dynamics (MD) simulations, we determined that CL molecules extensively engage with the paddle domain (PD) of mitochondrial fusion protein Optic Atrophy 1 (OPA1), which controls membrane-shaping mechanisms. Structure-function analysis confirmed the interactions between CL and two conserved motifs of OPA1 at the membrane-binding sites. We further developed a bromine-labeled CL probe to enhance cryoEM contrast and characterized the structure of OPA1 assemblies bound to the CL-brominated lipid bilayers. Our images provide direct evidence of CL enrichment within the OPA1-binding leaflet. Last, we observed a decrease in membrane remodeling activity for OPA1 in lipid compositions with increasing concentrations of monolyso-cardiolipin (MLCL). This suggests that the partial replacement of CL by MLCL, as observed in Barth syndrome-associated mutations of the tafazzin phospholipid transacylase, alters the malleability of the membrane and compromises proper remodeling. Together, these data provide insights into how biological membranes regulate the mechanisms governing mitochondrial homeostasis.

biophysics↗

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2023

RNA structures and dynamics with A resolution revealed by x-ray free electron lasers

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free electron laser sources to reveal the formation and ready identification of [A] scale features in structured and unstructured RNAs. New structural signatures of RNA secondary and tertiary structures are identified through wide angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base paired intermediate to assume a triple helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. In addition to understanding how RNA triplexes form and thereby function as dynamic signaling elements, this new method can vastly increase the rate of structure determination for these biologically essential, but mostly uncharacterized macromolecules.

biophysics↗

bioRxiv2023

Structural basis for functional properties of cytochrome c oxidase

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)4, we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

biophysics↗

bioRxiv2021

Membrane Constriction by ESCRT-III Proteins Induces Structural Lipid Bilayer Asymmetries

Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane remodeling system, we observed leaflet-level and protein-localized lipid structural patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates, and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated SDPC showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl (DHA) tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales. One-Sentence SummaryCryo-EM imaging of halogenated lipids and MD simulations provide molecular-scale insight into constricted bilayer structure.

biophysics↗

bioRxiv2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

bioRxiv2019

Membrane constriction and thinning by sequential ESCRT-III polymerization

The Endosomal Sorting Complexes Required for Transport (ESCRTs) mediate diverse membrane remodeling events. These activities typically require ESCRT-III proteins to stabilize negatively-curved membranes, although recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane curvature shaping by these proteins remains poorly understood. To learn how ESCRT-IIIs shape membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B+IST1, and IST1-only filaments by electron cryomicroscopy. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand upon the CHMP1B filament, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction, moreover, thins the underlying bilayer and lowers the barrier to membrane fission. Together, our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, tube constriction, and bilayer thinning.

biophysics↗