Biology subjects

Schmid, M. F.

Publications and source records attributed to Schmid, M. F..

17 recordsLinked to original sources

bioRxiv2026

Mechanism of the Sequential Trajectory of Allosteric Ring Closure in Human Chaperonin TRiC

Eukaryotic chaperonin TRiC assists in the folding of approximately 10% of the cytosolic proteome through an ATP-driven chamber closure cycle, yet the underlying closure mechanism has remained elusive. Here, using cryo-EM and deep learning analyses, we resolve, at near-atomic resolution, the temporal engagement sequence of the eight distinct subunits during human TRiC ring closure triggered by ATP binding. TRiC closes its chamber through the stepwise extension of an apical helical network among subunits around the ring, coordinated with sequential rotations of the equatorial domains. Each subunit responds differently to ATP, rendering the overall motion asymmetric and ordered. This work delineates the complete structural trajectory of allosteric propagation during ATP-binding-induced, rather than hydrolysis-induced, ring closure, and establishes a mechanistic framework linking ATP-driven conformational dynamics to substrate handling.

biophysics↗

bioRxiv2025

Cryogenic Electron Tomography Redefines Herpesvirus Capsid Assembly Intermediates Inside the Cell Nucleus.

Herpesviruses encapsulate their double-stranded DNA (dsDNA) genomes within an icosahedral nucleocapsid formed in the infected cell nucleus. Four biochemically purified nucleocapsids have been characterized, but their roles in herpesvirus replication remain controversial. The status of the capsid vertex-specific component (CVSC), essential for capsid stability and dsDNA packaging and retention, is also unclear. By integrating cryogenic focused ion beam milling with electron tomography and subtomogram averaging, we derived atomic models for all protein components, including the CVSC, across different herpesvirus capsid types within infected cell nuclei. Focused classification of pentonal vertex densities revealed differences in CVSC occupancy between genome-filled capsids and capsids lacking dsDNA, highlighting structural heterogeneity and providing insight into distinct capsid assembly stages in situ. These intra-nuclear findings redefine the maturation model of herpesvirus capsid assembly, advancing the understanding of herpesvirus replication, and demonstrate the effectiveness of in situ electron imaging by studying virus assembly within host cells.

microbiology↗

bioRxiv2025

Q-score as a reliability measure for protein, nucleic acid, and small molecule atomic coordinate models derived from 3DEM density maps

Atomic coordinate models are important in the interpretation of 3D maps produced with cryoEM and sub-tomogram averaging in cryoET, or more generically, 3D electron microscopy (3DEM). In addition to visual inspection of such maps and models, quantitative metrics convey the reliability of the atomic coordinates, in particular how well the model is supported by the experimentally determined 3DEM map. A recently introduced metric, Q-score, was shown to correlate well with the reported resolution of the map for well-fitted models. Here we present new statistical analyses of Q-scores based on its application to [~]10,000 maps and models archived in EMDB and PDB. Further we introduce two new metrics based on Q-score: Q-relative-all and Q-relative-resolution to compare a map and model to all entries in the EMDB and those with similar resolution respectively. We also explore through illustrative examples of proteins, nucleic acids, and small molecules how Q-scores can indicate whether the atomic coordinates are well-fitted to 3DEM maps and whether some parts of a map may be poorly resolved due to factors such as molecular flexibility, radiation damage, and/or conformational heterogeneity. Lastly, we show examples of how Q-scores can effectively be converted to atomic B-factors. These analyses provide a basis for how Q-scores can be interpreted effectively to evaluate 3DEM maps and atomic coordinate models prior to publication and archiving. SynopsisQ-scores are calculated each atom in models fitted to 3DEM (3D electron microscopy) maps. They measure how well the model fits the map, and also reflect the quality of the map as they correlate to resolution. Here we develop a statistical model for Q-scores applied to many maps and models in the EMDB (Electron Microscopy Database) and PDB (Protein Data Bank) respectively, and show how it can be used to assess the reliability of entire models as well as their subcomponents.

biochemistry↗

bioRxiv2024

Visualizing nucleation, condensation and propagation of β-tubulin folding in chaperonin TRiC

The folding nucleus (FN) initiates protein folding and enables an efficient folding pathway. Here we directly visualize the tubulin FN consisting of a nonnative, partially assembled Rossmann fold, in the closed chamber of human chaperonin TRiC. Chaperonin TRiC interacts with non-natively folded secondary structural elements, stabilizing the nucleus for transition into its first native domain. Through progressive folding, the unfolded sequence goes through drastic spatial arrangement in the TRiC chamber to sample the conformational space, mediated by the highly dynamic CCT tails. The observed presence of individual nonnative secondary structures first in the nonnative FN and then around the incrementally folded native domains supports the hypothesis that tubulin folding in TRiC is a hierarchical process of nucleation, condensation and propagation in cooperation with TRiC subunits.

biophysics↗

bioRxiv2024

Missing Wedge Completion via Unsupervised Learning with Coordinate Networks

Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data collection angles. Recently, supervised deep learning methods leveraging convolutional neural networks (CNNs) have considerably addressed this issue; however, their pretraining requirements render them susceptible to inaccuracies and artifacts, particularly when representative training data is scarce. To overcome these limitations, we introduce a proof-of-concept unsupervised learning approach using coordinate networks (CNs) that optimizes network weights directly against input projections. This eliminates the need for pretraining, reducing reconstruction runtime by 3 - 20x compared to supervised methods. Our in silico results show improved shape completion and reduction of missing wedge artifacts, assessed through several voxel-based image quality metrics in real space and a novel directional Fourier Shell Correlation (FSC) metric. Our study illuminates benefits and considerations of both supervised and unsupervised approaches, guiding the development of improved reconstruction strategies.

molecular biology↗

bioRxiv2024

Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for [~]50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cells apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cells affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

plant biology↗

bioRxiv2024

Cost-benefit analysis of cryogenic electron tomography subtomogram averaging of chaperonin MmCpn at near atomic resolution.

Cryogenic electron microscopy single particle reconstruction (cryoEM-SPR) has evolved into a routine approach for determining macromolecule structures to near-atomic resolution. Cryogenic electron tomography subtomogram averaging (cryoET-STA) towards similar resolution, in contrast, is still under active development. CryoET can capture the 3D snapshot of individual macromolecules by stage tilting, offering multiple angular views per particle than the single particle reconstruction approach. Here we use the archaea chaperonin MmCpn as a model macromolecule to investigate the resolution limiting factors of cryoET-STA in terms of cumulative electron dose, ice thickness, subtomogram numbers and tilt angle ranges. By quantitative analysis of these factors against the STA reconstruction resolution, we delineate the feasibility of attaining high resolution structure determination with cryoET-STA. This study provides biophysical guidance for the application of cryoET-STA towards high resolution and the cost against benefit of using cryoET-STA to achieve an efficient outcome at the desired resolution.

biophysics↗

bioRxiv2023

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy an optimized cryo-ET platform that enables cellular-structural biology in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using this platform, we reconstructed sub-nanometer resolution structures of the human cardiac muscle thin filament, a central component of the contractile machinery. Reconstructing the troponin complex, a regulatory component of the thin filament, from within cells, we identified previously unobserved conformations that highlight the structural flexibility of this regulatory complex. We next measured the impact of chemical and genetic perturbations associated with cardiovascular disease on the structure of troponin. In both cases, we found changes in troponin structure that are consistent with known disease phenotypes--highlighting the value of our approach for dissecting complex disease mechanisms in the cellular context.

biophysics↗

bioRxiv2022

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. In this paper, we perform cryogenic electron tomography (cryo-ET) on isolated merozoites to visualize the apical machinery. Through tomography reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry-secretory-apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly patterning. These results provide new insight into the essential features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells.

cell biology↗

bioRxiv2022

CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

Huntingtons Disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. Patient-derived induced pluripotent stem cells (iPSCs) can help understand disease; however, defining pathological biomarkers is challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered mitochondria with enlarged granules and distorted cristae, and thin sheet aggregates in double membrane-bound organelles. We used artificial intelligence to quantify mitochondrial granules, and proteomics to show differential protein content in HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC-neurons and reduced phenotypes in BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

neuroscience↗

bioRxiv2021

In situ Alphavirus Assembly and Budding Mechanism Revealed by Cellular CryoET

Chikungunya virus (CHIKV) is an alphavirus and the etiological agent for debilitating arthritogenic disease in humans. Previous studies with purified virions or budding mutants have not resolved the structural mechanism of alphavirus assembly in situ. Here we used cryogenic electron tomography (cryoET) imaging of CHIKV-infected human cells and subvolume classification to resolve distinct assembly intermediate conformations. These structures revealed that particle formation is driven by the spike envelope layer. Additionally, we showed that asymmetric immature nucleocapsids (NCs) provide scaffolds to trigger assembly of the icosahedral spike lattice, which progressively transforms immature NCs into icosahedral cores during virus budding. Further, cryoET of the infected cells treated with neutralizing antibodies (NAbs) showed that NAb-induced blockage of CHIKV assembly was achieved by preventing spike-spike lateral interactions that are required to bend the plasma membrane around NC cores. These findings provide molecular mechanisms for designing antivirals targeting spike-driven assembly/budding of viruses.

microbiology↗

bioRxiv2021

Cryo-ET reveals two major tubulin-based cytoskeleton structures in Toxoplasma gondii

In the obligate intracellular parasite, Toxoplasma gondii, the subpellicular microtubules (SPMTs) help maintain shape, while the apical conoid (also tubulin-based) is implicated in invasion. Here, we use cryo-electron tomography to determine the molecular structures of the SPMTs and the conoid-fibrils (CFs) in vitrified and detergent-lysed parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intraluminal spiral (IS) lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasites long axis. Each CF is composed of 9 tubulin protofilaments, that produce a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism, illustrates the versatility of these critical structures.

molecular biology↗

bioRxiv2020

Resolving Individual-Atom of Protein Complex using Commonly Available 300-kV Cryo-electron Microscopes

Breakthroughs in single-particle cryo-electron microscopy (cryo-EM) technology have made near-atomic resolution structure determination possible. Here, we report a [~]1.35-[A] structure of apoferritin reconstructed from images recorded on a Gatan K3 or a Thermo Fisher Falcon 4 detector in a commonly available 300-kV Titan Krios microscope (G3i) equipped with or without a Gatan post-column energy filter. Our results demonstrate that the atomic-resolution structure determination can be achieved by single-particle cryo-EM with a fraction of a day of automated data collection. These structures resolve unambiguously each heavy atom (C, N, O, and S) in the amino acid side chains with an indication of hydrogen atoms presence and position, as well as the unambiguous existence of multiple rotameric configurations for some residues. We also develop a statistical and chemical based protocol to assess the positions of the water molecules directly from the cryo-EM map. In addition, we have introduced a B factor equivalent to the conventional B factor traditionally used by crystallography to annotate the atomic resolution model for determined structures. Our findings will be of immense interest among protein and medicinal scientists engaging in both basic and translational research.

biophysics↗

bioRxiv2020

A 3.4-A cryo-EM structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles

Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV and SARS-CoV-2. Here we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-[A] resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a 3-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other amino acid positions as well as differences in glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies, or drugs, in the native state of the coronavirus particles.

biophysics↗

bioRxiv2020

Outcomes of the 2019 EMDataResource model challenge: validation of cryo-EM models at near-atomic resolution

This paper describes outcomes of the 2019 Cryo-EM Map-based Model Metrics Challenge sponsored by EMDataResource (www.emdataresource.org). The goals of this challenge were (1) to assess the quality of models that can be produced using current modeling software, (2) to check the reproducibility of modeling results from different software developers and users, and (3) compare the performance of current metrics used for evaluation of models. The focus was on near-atomic resolution maps with an innovative twist: three of four target maps formed a resolution series (1.8 to 3.1 [A]) from the same specimen and imaging experiment. Tools developed in previous challenges were expanded for managing, visualizing and analyzing the 63 submitted coordinate models, and several novel metrics were introduced. The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual laboratory experiments and holdings of structure data archives such as the Protein Data Bank. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived from these benchmark maps by 13 participating teams, representing both widely used and novel modeling approaches. We also evaluate the pros and cons of the commonly used metrics to assess model quality and recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed density in the cryo-EM map.

biochemistry↗

bioRxiv2019

Measurement of Atom Resolvability in CryoEM Maps with Q-scores

CryoEM density maps are now at the point where resolvability of individual atoms can be achieved. However, resolvability is not necessarily uniform throughout the map. We introduce a quantitative parameter to characterize the resolvability of individual atoms in cryoEM maps, the map Q-score. Q-scores can be calculated for atoms in proteins, nucleic acids, water, ligands, and other solvent atoms, using models fitted to or derived from cryoEM maps. Q-scores can also be averaged to represent larger features such as entire residues and nucleotides. Averaged over entire models, Q-scores correlate very well with the estimated resolution of cryoEM maps for both protein and RNA. Assuming the models they are calculated from are well-fitted to the map, Q-scores can thus be used as another measure to indicate resolvability of features in cryoEM maps at various scales, from entire complexes down to individual atoms. Q-score analysis of multiple cryoEM maps of the same proteins derived from different labs confirms reproducibility of structural features down to water and ion atoms.

biochemistry↗

bioRxiv2017

Defining the Layers of a Sensory Cilium with STORM and Cryo-Electron Nanoscopies

Primary cilia are cylindrical organelles extending from the surface of most animal cells that have been implicated in a host of signaling and sensory functions. Genetic defects in their component molecules, known as \"ciliopathies\" give rise to devastating symptoms, ranging from defective development, to kidney disease, to progressive blindness. The detailed structures of these organelles and the true functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We describe the combination of cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of the mammalian retina. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of these compartments, including Ca2+-binding protein centrin-2 in the lumen of the axoneme, acetylated tubulin forming the axoneme, the glycocalyx extending outward from the surface of the plasma membrane, and molecular residents of the space between axoneme and ciliary membrane, including Arl13B, intraflagellar transport proteins, BBS5, and syntaxin-3. Within this framework we document that deficiencies in the ciliopathy proteins BBS2, BBS7 and BBS9 lead to inappropriate accumulation of proteins in rod outer segments while largely preserving their sub-domain localization within the connecting cilium region, but alter the distribution of syntaxin-3 clusters.

cell biology↗