Most downloaded biology preprints, all time
in category biophysics
6,088 results found. For more information, click each entry to expand.
26,073 downloads bioRxiv biophysics
Sara Linse, Tom Scheidt, Katja Bernfur, Michele Vendruscolo, Christopher M Dobson, Samuel I. A. Cohen, Eimantas Sileikis, Martin Lundquist, Fang Qian, Tiernan O’Malley, Thierry Bussiere, Paul H Weinreb, Catherine K Xu, Georg Meisl, Sean R. A. Devenish, Tuomas Knowles, Oskar Hansson
The amyloid cascade hypothesis, according to which the self-assembly of amyloid-β peptide (Aβ) is a causative process in Alzheimer’s disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aβ-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. We bring together kinetic analysis with quantitative binding measurements to address the mechanisms of action of four clinical stage anti-Aβ antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We reveal and quantify the striking differences of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Our results uncover that, uniquely amongst these four antibodies, aducanumab dramatically reduces the flux of oligomeric forms of Aβ.
20,266 downloads bioRxiv biophysics
Single particle cryo-EM is a powerful method to solve the three-dimensional structures of biological macromolecules. The technological development of electron microscopes, detectors, automated procedures in combination with user friendly image processing software and ever-increasing computational power have made cryo-EM a successful and largely expanding technology over the last decade. At resolutions better than 4 Å , atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher (< 1.5 Å ) resolution, which so far could not be attained by cryo-EM. The direct visualization of atom positions is essential for understanding protein-catalyzed chemical reaction mechanisms and to study drug- binding and -interference with protein function. Here we report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution). For the first time in cryo-EM we can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications. Beyond the nominal improvement in resolution we can also show a significant improvement in quality of the cryo-EM density map which is highly relevant for using cryo-EM in structure-based drug design. ### Competing Interest Statement The authors have declared no competing interest.
15,517 downloads bioRxiv biophysics
Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M.G.E. Brown, Ioana T. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W Hardwick, Dimitri Y Chirgadze, Garib Murshudov, A. Radu Aricescu, Sjors Scheres
The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. ### Competing Interest Statement A.K., S.M., L.Y., D.K., E.V.P., E.d.J., J.K., M.B., J.M., and P.T are employees of Thermo Fisher Scientific.
13,208 downloads bioRxiv biophysics
Magnetoreception, the perception of the geomagnetic field, is a sensory modality well-established across all major groups of vertebrates and some invertebrates, but its presence in humans has been tested rarely, yielding inconclusive results. We report here a strong, specific human brain response to ecologically-relevant rotations of Earth-strength magnetic fields. Following geomagnetic stimulation, a drop in amplitude of EEG alpha oscillations (8-13 Hz) occurred in a repeatable manner. Termed alpha event-related desynchronization (alpha-ERD), such a response is associated with sensory and cognitive processing of external stimuli. Biophysical tests showed that the neural response was sensitive to the dynamic components and axial alignment of the field but also to the static components and polarity of the field. This pattern of results implicates ferromagnetism as the biophysical basis for the sensory transduction and provides a basis to start the behavioral exploration of human magnetoreception.
10,961 downloads bioRxiv biophysics
A novel and highly pathogenic coronavirus (2019-nCoV) has caused an outbreak in Wuhan city, Hubei province of China since December 2019, and soon spread nationwide and spilled over to other countries around the world. To better understand the initial step of infection at atomic-level, we determined the crystal structure of the 2019-nCoV spike receptor-binding domain (RBD) bound with the cell receptor ACE2 at 2.45 angstrom resolution. The overall ACE2-binding mode of the 2019-nCoV RBD is nearly identical to that of the SARS-CoV RBD, which also utilizes ACE2 as the cell receptor. Structural analysis identified residues in 2019-nCoV RBD critical for ACE2 binding, and majority of which are either highly conserved or shared similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly argue for a convergent evolution between 2019-nCoV and SARS-CoV RBD for improved binding to ACE2 despite of being segregated in different genetic lineages in the betacoronavirus genus. The epitopes of two SARS-CoV antibodies targeting the RBD are also analyzed with the 2019-nCoV RBD, providing insights into future identification of cross-reactive antibodies.
10,081 downloads bioRxiv biophysics
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma. 3a has been implicated in viral release, inhibition of autophagy, inflammasome activation, and cell death and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 [A] resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.
9,982 downloads bioRxiv biophysics
Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher-resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.
9,846 downloads bioRxiv biophysics
Beginning in late 2016, diplomats posted to the United States embassy in Cuba began to experience unexplained health problems including ear pain, tinnitus, vertigo, and cognitive difficulties which reportedly began after they heard strange noises in their homes or hotel rooms. In response, the U.S. government dramatically reduced the number of diplomats posted at the U.S. embassy in Havana. U.S. officials initially believed a sonic attack might be responsible for their ailments. The sound linked to these attacks, which has been described as a high-pitched beam of sound, was recorded by U.S. personnel in Cuba and released by the Associated Press (AP). Because these recordings are the only available non-medical evidence of the sonic attacks, much attention has focused on identifying health problems and the origin of the acoustic signal. As shown here, the calling song of the Indies short-tailed cricket (Anurogryllus celerinictus) matches, in nuanced detail, the AP recording in duration, pulse repetition rate, power spectrum, pulse rate stability, and oscillations per pulse. The AP recording also exhibits frequency decay in individual pulses, a distinct acoustic signature of cricket sound production. While the temporal pulse structure in the recording is unlike any natural insect source, when the cricket call is played on a loudspeaker and recorded indoors, the interaction of reflected sound pulses yields a sound virtually indistinguishable from the AP sample. This provides strong evidence that an echoing cricket call, rather than a sonic attack or other technological device, is responsible for the sound in the released recording. Although the causes of the health problems reported by embassy personnel are beyond the scope of this paper, our findings highlight the need for more rigorous research into the source of these ailments, including the potential psychogenic effects, as well as possible physiological explanations unrelated to sonic attacks.
9,230 downloads bioRxiv 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.
8,740 downloads bioRxiv 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.
8,449 downloads bioRxiv biophysics
Rapidly spreading SARS-CoV-2 variants present not only an increased threat to human health due to the confirmed greater transmissibility of several of these new strains but, due to conformational changes induced by the mutations, may render first-wave SARS-CoV-2 convalescent sera, vaccine-induced antibodies, or recombinant neutralizing antibodies (nAbs)ineffective. To be able to assess the risk of viral escape from neutralization by first-wave antibodies, we leveraged our capability for Molecular Dynamic (MD) simulation of the spike receptor binding domain (S RBD)and its binding to human angiotensin-converting enzyme 2 (hACE2) to predict alterations in molecular interactions resulting from the presence of the E484K, K417N, and N501Y variants found in the South African 501Y.V2 strain - alone and in combination. We report here the combination of E484K, K417N, and N501Y results in the highest degree of conformational alterations of S RBD when bound to hACE2, compared to either E484K or N501Y alone. Both E484K and N501Y increase affinity of S RBD for hACE2 and E484K in particular switched the charge on the flexible loop region of S RBD which leads to the formation of novel favorable contacts. Enhanced affinity of S RBD for hACE2 very likely underpins the greater transmissibility conferred by the presence of either E484K or N501Y; while the induction of conformational changes may provide an explanation for evidence that the 501Y.V2 variant, distinguished from the B.1.1.7 UK variant by the presence of E484K, is able to escape neutralization by existing first-wave anti-SARS-CoV-2 antibodies and re-infect COVID-19 convalescent individuals.
8,004 downloads bioRxiv biophysics
The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens is currently largely uncoupled from subsequent data evaluation, correction and processing. Therefore, the acquisition strategy is difficult to optimize during data collection, often leading to suboptimal microscope usage and disappointing results. Here we provide Warp, a software for real-time evaluation, correction, and processing of cryo-EM data during their acquisition. Warp evaluates and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. Warp also rapidly corrects micrographs for global and local motion, and estimates the local defocus with the use of novel algorithms. The software further includes a deep learning-based particle picking algorithm that rivals human accuracy to make the pre-processing pipeline truly automated. The output from Warp can be directly fed into established tools for particle classification and 3D image reconstruction. In a benchmarking study we show that Warp automatically processed a published cryo-EM data set for influenza virus hemagglutinin, leading to an improvement of the nominal resolution from 3.9 Å to 3.2 Å. Warp is easy to install, computationally inexpensive, and has an intuitive and streamlined user interface.
7,789 downloads bioRxiv biophysics
The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles' that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19). ### Competing Interest Statement The authors have declared no competing interest.
7,286 downloads bioRxiv biophysics
Correction of electron beam-induced sample motion is one of the major factors contributing to the recent resolution breakthroughs in cryo-electron microscopy. Improving the accuracy and efficiency of motion correction can lead to further resolution improvement. Based on observations that the electron beam induces doming of the thin vitreous ice layer, we developed an algorithm to correct anisotropic image motion at the single pixel level across the whole frame, suitable for both single particle and tomographic images. Iterative, patch-based motion detection is combined with spatial and temporal constraints and dose weighting. The multi-GPU accelerated program, MotionCor2, is sufficiently fast to keep up with automated data collection. The result is an exceptionally robust strategy that can work on a wide range of data sets, including those very close to focus or with very short integration times, obviating the need for particle polishing. Application significantly improves Thon ring quality and 3D reconstruction resolution.
7,202 downloads bioRxiv biophysics
Maxwell I. Zimmerman, Justin R. Porter, Michael D Ward, Sukrit Singh, Neha Vithani, Artur Meller, Upasana L. Mallimadugula, Catherine E. Kuhn, Jonathan H. Borowsky, Rafal Wiewiora, Matthew F. D. Hurley, Aoife M Harbison, Carl A. Fogarty, Joseph E. Coffland, Elisa Fadda, Vincent A. Voelz, John D. Chodera, Gregory R. Bowman
SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of "cryptic" epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 "cryptic" pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas. ### Competing Interest Statement The authors have declared no competing interest.
7,156 downloads bioRxiv 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.
6,784 downloads bioRxiv biophysics
Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ . In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M , a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 Å. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level. ### Competing Interest Statement The authors have declared no competing interest.
6,453 downloads bioRxiv biophysics
Technical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 Å resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.
6,212 downloads bioRxiv biophysics
RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Motivated by the need for stabilized COVID-19 mRNA vaccines, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall rate of hydrolysis. To characterize the stabilization achievable through structure design, we compare optimization of AUP by conventional mRNA design methods to results from the LinearDesign algorithm, a new Monte Carlo tree search algorithm called RiboTree, and crowdsourcing through the OpenVaccine challenge on the Eterna platform. Tests were carried out on mRNAs encoding nanoluciferase, green fluorescent protein, and COVID-19 mRNA vaccine candidates encoding SARS-CoV-2 epitopes, spike receptor binding domain, and full-length spike protein. We find that Eterna and RiboTree significantly lower AUP while maintaining a large diversity of sequence and structure features that correlate with translation, biophysical size, and immunogenicity. Our results suggest that increases in in vitro mRNA half-life by at least two-fold are immediately achievable and that further stability improvements may be enabled with thorough experimental characterization of RNA hydrolysis. ### Competing Interest Statement The authors have declared no competing interest.
6,206 downloads bioRxiv biophysics
The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 15,000,000 infections and 600,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates the host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viruses, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans, and the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike’s receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift towards the “down” state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which may be exploited by therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development. ### Competing Interest Statement The authors have declared no competing interest.
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