Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 67,095 bioRxiv papers from 295,270 authors.
Most downloaded bioRxiv papers, since beginning of last month
in category biophysics
2,778 results found. For more information, click each entry to expand.
24,776 downloads biophysics
Sara Linse, Tom Scheidt, Katja Bernfur, Michele Vendruscolo, Christopher M. Dobson, Samuel IA Cohen, Eimantas Sileikis, Martin Lundquist, Fang Qian, Tiernan O'Malley, Thierry Bussiere, Paul H Weinreb, Catherine K Xu, Georg Meisl, Sean Devenish, Tuomas PJ Knowles, Oskar Hansson
Alzheimer's disease affects nearly 50 million people worldwide with an overall cost of over 1% of the global economy. The amyloid cascade hypothesis, according to which the misfolding and aggregation of the amyloid-β peptide (Aβ) triggers a series of pathological processes that eventually result in massive brain tissue loss, has driven many therapeutic efforts for the past 20 years. Repeated failures, however, have highlighted the challenges of characterizing the molecular mechanisms of therapeutic candidates targeting Aβ, and connecting them to the outcomes of clinical trials. Here, we determine the mechanism of action of four clinical stage antibodies (aducanumab, gantenerumab, bapineuzumab and solanezumab). We quantify the dramatic differences that these antibodies have on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for the monomeric and fibrillar forms of Aβ. We show that the binding parameters of each antibody correlate with the corresponding level of amyloid clearance in clinical trials and that the reduction in oligomer flux correlates with the cognitive improvement. We reveal that, uniquely amongst these four antibodies, aducanumab dramatically reduces the flux of oligomeric forms of Aβ. These results demonstrate the power of quantitative molecular analysis in predicting the outcomes of clinical trials.
1,745 downloads biophysics
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and pathogenicity.
983 downloads biophysics
Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids, aimed at preserving thin, uniform vitrified ice and improving protein adsorption, have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6 angstrom resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipo-proteins by at least five times, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, our approach allows for customized grid designs targeting specific proteins, due to its broad compatibility with a variety of nanomaterials.
648 downloads biophysics
Quantitative imaging of biological architecture with fluorescent labels is not as scalable as genomic or proteomic measurements. Here, we combine quantitative label-free imaging and deep neural networks for scalable analysis of complex structures. We reconstruct quantitative three-dimensional density, anisotropy, and orientation in live cells and tissue slices from polarization- and depth-resolved images. We report a computationally efficient variant of U-Net architecture that predicts a 3D fluorescent structure from its morphology and physical properties. We evaluate the performance of our models by predicting F-actin and nuclei in mouse kidney tissue. Further, we report label-free imaging of axon tracts and predict level of myelination in human brain tissue sections. We demonstrate the model's ability to rescue inconsistent labeling. We anticipate that the proposed approach will enable quantitative analysis of architectural order across scales of organelles to tissues.
612 downloads biophysics
Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Forster resonance energy transfer (FRET) to quantify molecular interactions. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.
596 downloads biophysics
Deep neural networks have recently enabled spectacular progress in predicting protein structures, as demonstrated by DeepMin’s winning entry with Alphalfold at the latest Critical Assessment, of Structure Prediction competition (CASP13). The best protein prediction pipeline leverages intermolecular distance predictions to assemble a final protein model, but this distance prediction network has not been published. Here, we make a trained implementation of this network available to the broader scientific community. We also benchmark its predictive power in the related task of contact prediction against the CASP13 contact prediction winner TripletRes. Access to ProSPr will enable other labs to build on best in class protein distance predictions and to engineer superior protein reconstruction methods.
583 downloads biophysics
Estimates of heat transfer rates during plunge cooling and patterns of ice observed in cryoEM samples indicate that the grid bars cool much more slowly than do the support foil and sample near the middle of the grid openings. The resulting transient temperature differences generate transient tensile stresses in the support foil. Most of this foil stress develops while the sample is liquid and cooling toward its glass transition Tg, and so does not generate tensile sample stress. As the grid bars continue cooling toward the cryogen temperature and contracting, the tensile stress in the foil is released, placing the sample in compressive stress. Radiation-induced creep in the presence of this compressive stress should generate a doming of the sample in the foil openings, as is observed experimentally. Crude estimates of the magnitude of doming that may be generated by this mechanism are consistent with observation. Several approaches to reducing beam-induced motion are discussed.
561 downloads biophysics
Bardet-Biedl syndrome (BBS) is an incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a master regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the b-propeller domain of BBS1, the key subunit implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis predominantly results from disruption of autoinhibition and activation.
543 downloads biophysics
HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4,5) bisphosphate, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T cells. Analysis of HIV-1 infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.
541 downloads biophysics
At the initial stage of the cryo-electron microcopy (cryo-EM) samples irradiated by electrons, the cryo-EM samples suffer from a rapid "burst phase" (first 3~4 e-/Å2) of beam induced motion (BIM) which is too fast to be corrected by existing motion correction software, and lowers the quality of the initial frames. Therefore, these least radiation damaged, but ruined frames are commonly excluded or down-weighted during data processing, which reduces the undamaged signals in the reconstruction and decreases the reconstruction resolution by weakening the averaging power. Here, we show that increasing the freezing temperature of cryo-EM samples suppresses the BIM in this phase. The quality of initial frames is partially recovered after BIM correction and is better than that of subsequent frames in certain frames. Incorporating these initial frames into the reconstruction increases the resolution, at an equivalent of ~60% extra data. Moreover, these frames are least radiation damaged, thus preserves the high quality cryo-EM density of radiation sensitive residues. Such density is usually damaged or very weak in the canonical 3D reconstruction. In addition, we found that a different kind of radiation damage neglected previously occurs in the per-frame reconstruction after the exposure of 2.5 e-/Å2. Such radiation damage distorts the density of atoms. The deformation can be avoided by only including the frames from the first 2.5 e-/Å2 into the reconstruction. Overall, the high temperature freezing not only provides extra undamaged signal to the reconstruction, but also increases the resolution of the reconstruction.
392 downloads biophysics
The ultimate goal of biological superresolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here, we show that, by localizing individual switchable fluorophores with a probing doughnut-shaped excitation beam, MINFLUX nanoscopy provides 1 to 3 nanometer resolution in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing doughnut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than popular camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.
366 downloads biophysics
The number of structures and molecular dynamics simulations of proteins is exploding owing to dramatic advances in cryo-electron microscopy, crystallography, and computing. One of the most powerful ways to analyze structural information involves comparisons of interatomic interactions across different structures or simulations of the same protein or related proteins from the same family (e.g. different GPCRs). Such comparative analyses are of interest to a wide range of researchers but currently prove challenging for all but a few. To facilitate comparative structural analyses, we have developed tools for (i) rapidly computing and comparing interatomic interactions and (ii) interactively visualizing interactions to enable structure-based interpretations. Using these tools, we have developed the Contact Comparison Atlas, a web-based resource for the comparative analysis of interactions in structures and simulations of proteins. Using the Contact Comparison Atlas and our tools, we have identified patterns of interactions with functional implications in structures of G-protein-coupled receptors, G proteins and kinases and in the dynamics of muscarinic receptors. The Contact Comparison Atlas can be used to enable structure modeling, drug discovery, protein engineering, and the prediction of disease-associated mutations.
360 downloads biophysics
Zachary T Berndsen, Srirupa Chakraborty, Xiaoning Wang, Christopher A Cottrell, Jonathan L Torres, Jolene K. Diedrich, Cesar A. López, John R. Yates, Marit J van-Gils, James C. Paulson, S. Gnanakaran, Andrew B Ward
The dense array of N-linked glycans on the HIV-1 Envelope Glycoprotein (Env), known as the "glycan shield", is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM) and computational modeling to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of inter-glycan interactions and drive higher-order structuring within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and provides a mapping of potentially immunogenic sites on Env. Analysis of the same Env across a range of glycosylation states revealed that subtle changes in glycan occupancy, composition, and dynamics can impact glycan shield structure and epitope accessibility. We also performed site-specific mass-spectrometry analysis on the same samples and show how cryo-EM can complement such studies. Finally, we found that highly connected glycan sub-domains are resistant to enzymatic digestion and help stabilize the pre-fusion trimer state, suggesting functionality beyond immune evasion.
340 downloads biophysics
Cell stiffness is a key cellular material property that changes locally and temporally during many cellular functions including migration, adhesion, and growth. Currently, it is widely accepted that cells adapt their mechanical properties to the stiffness of their surroundings. The link between cortical cell stiffness and substrate mechanics was hypothesized based on atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates. Here we show that the force applied by AFM can result in a significant deformation not only of the cell surface but also of the underlying substrate if it is sufficiently soft. This 'soft substrate effect' leads to an underestimation of a cell's elastic modulus on substrates softer than the cells when fitting the indentation data using a standard Hertz model, as confirmed by finite element modelling (FEM) and AFM measurements of calibrated polyacrylamide beads, microglial cells, and fibroblasts. To account for this substrate deformation, we developed the 'composite cell-substrate model' ('CoCS' model), which does not require any knowledge about the cell-substrate geometry, and which can be implemented in any standard AFM indentation measurement. Our results provide a new formalism to analyze indentation data obtained for cells cultured on soft matrices, and they suggest that cortical cell stiffness is largely independent of substrate mechanics, which has significant implications for our interpretation of many physiological and pathological processes.
306 downloads biophysics
Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.
298 downloads biophysics
Wanchao Yin, Zhihai Li, Mingliang Jin, Yuling Yin, Parker W. de Waal, Kuntal Pal, Yanting Yin, Xiang Gao, Yuanzheng He, Jing Gao, Xiaoxi Wang, Yan Zhang, Hu Zhou, Karsten Melcher, Yi Jiang, Yao Cong, Edward Zhou, Xuekui Yu, H. Eric Xu
Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are β-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin-rhodopsin complex by a 90° rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2‒NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2‒NTSR1 complex structure may provide an alternative template for modeling arrestin-GPCR interactions.
289 downloads biophysics
Alexandra Zak, Sara Violeta Merino-Cortès, Anaïs Sadoun, Avin Babataheri, Stéphanie Dogniaux, Sophie Dupré-Crochet, Elodie Hudik, Hai-Tao He, Abdul I Barakat, Yolanda R Carrasco, Yannick Hamon, Pierre-Henri Puech, Claire Hivroz, Oliver Nüsse, Julien Husson
To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. Using an innovative micropipette rheometer, we show in three different cell types that when stimulated by microbeads mimicking target cells, leukocytes become up to ten times stiffer and more viscous. These mechanical changes initiate within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. The current results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new way to investigate cell mechanical processes. Our findings also suggest that dynamic immuno-mechanical measurements provide an identifier of leuko-cyte type and an indicator of the cell's state of activation.
288 downloads biophysics
We present an implementation of the Gillespie algorithm that simulates the stochastic kinetics of nascent and mature RNA. Our model includes two-state gene regulation, RNA synthesis initiation and stepwise elongation, release to the cytoplasm, and stepwise degradation, a granular description currently tractable only by simulation. To facilitate comparison with experimental data, the algorithm predicts fluorescent probe signals measurable by single-cell RNA imaging. We approach the inverse problem of estimating underlying parameters in a five-dimensional parameter space and suggest optimization heuristics that successfully recover known reaction rates from simulated gene expression turn-on data. The simulation framework includes a graphical user interface, available as a MATLAB app at https://data.caltech.edu/records/1287.
280 downloads biophysics
We present methods that detect three types of aberrations in single-particle cryo-EM datasets: symmetrical and antisymmetrical optical aberrations and magnification anisotropy. Because our methods only depend on the availability of a preliminary 3D reconstruction from the data, they can be used to correct for these aberrations for any given cryo-EM data set, a posteriori. Using five publicly available data sets, we show that considering these aberrations improves the resolution of the 3D reconstruction when the effects are present. The methods are implemented in version 3.1 of our open-source software package RELION.
277 downloads biophysics
Understanding the spatial organisation of the genome in the cell nucleus is one of the current grand challenges in biophysics. Certain biochemical -- or epigenetic -- marks that are deposited along the genome are thought to play an important, yet poorly understood, role in determining genome organisation and cell identity. The physical principles underlying the interplay between epigenetic dynamics and genome folding remain elusive. Here we propose and study a theory that assumes a coupling between epigenetic mark and genome densities, and which can be applied at the scale of the whole nucleus. We show that equilibrium models are not compatible with experiments and a qualitative agreement is recovered by accounting for non-equilibrium processes which can stabilise microphase separated epigenomic domains. We finally discuss the potential biophysical origin of these terms.
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