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Rxivist combines biology preprints from bioRxiv and medRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 212,235 papers from 844,985 authors.

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in category biophysics

8,105 results found. For more information, click each entry to expand.

1: Kinetic fingerprints differentiate anti-Aβ therapies
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Posted 22 Oct 2019

Kinetic fingerprints differentiate anti-Aβ therapies
26,211 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.

2: Breaking the next Cryo-EM resolution barrier - Atomic resolution determination of proteins!
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Posted 22 May 2020

Breaking the next Cryo-EM resolution barrier - Atomic resolution determination of proteins!
21,454 downloads bioRxiv biophysics

Ka Man Yip, Niels Fischer, Elham Paknia, Ashwin Chari, Holger Stark

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.

3: Single-particle cryo-EM at atomic resolution
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Posted 22 May 2020

Single-particle cryo-EM at atomic resolution
16,841 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 HW 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.

4: Transduction of the Geomagnetic Field as Evidenced from Alpha-band Activity in the Human Brain
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Posted 20 Oct 2018

Transduction of the Geomagnetic Field as Evidenced from Alpha-band Activity in the Human Brain
14,850 downloads bioRxiv biophysics

Connie X Wang, Isaac A Hilburn, Daw-An Wu, Yuki Mizuhara, Christopher P Cousté, Jacob N. H. Abrahams, Sam E Bernstein, Ayumu Matani, Shinsuke Shimojo, Joseph L Kirschvink

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.

5: Robust deep learning based protein sequence design using ProteinMPNN
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Posted 04 Jun 2022

Robust deep learning based protein sequence design using ProteinMPNN
12,706 downloads bioRxiv biophysics

Justas Dauparas, Ivan Anishchenko, Nathaniel Bennett, Hua Bai, Robert J Ragotte, Lukas F Milles, Basile I M Wicky, Alexis Courbet, Robbert J. de Haas, Neville P Bethel, Philip J. Y. Leung, Timothy F. Huddy, Sam Pellock, Doug Tischer, Frederick Chan, Brian Koepnick, Hannah Nguyen, Alex Kang, Banumathi Sankaran, Asim K Bera, Neil P King, David Baker

While deep learning has revolutionized protein structure prediction, almost all experimentally characterized de novo protein designs have been generated using physically based approaches such as Rosetta. Here we describe a deep learning based protein sequence design method, ProteinMPNN, with outstanding performance in both in silico and experimental tests. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4%, compared to 32.9% for Rosetta. Incorporation of noise during training improves sequence recovery on protein structure models, and produces sequences which more robustly encode their structures as assessed using structure prediction algorithms. We demonstrate the broad utility and high accuracy of ProteinMPNN using X-ray crystallography, cryoEM and functional studies by rescuing previously failed designs, made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target binding proteins.

6: Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs
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Posted 18 Jun 2020

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs
11,494 downloads bioRxiv biophysics

David M Kern, Em Sorum, Sonali Mali, Christopher M. Hoel, Savitha Sridharan, Jonathan P Remis, Daniel B Toso, Abhay Kotecha, Diana M Bautista, Stephen G Brohawn

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.

7: Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor
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Posted 20 Feb 2020

Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor
11,464 downloads bioRxiv biophysics

Jun Lan, Jiwan Ge, Jinfang Yu, Sisi Shan, Huan Zhou, Shilong Fan, Qi Zhang, Xuanling Shi, Qisheng Wang, Linqi Zhang, Xinquan Wang

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.

8: A structural biology community assessment of AlphaFold 2 applications
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Posted 26 Sep 2021

A structural biology community assessment of AlphaFold 2 applications
11,131 downloads bioRxiv biophysics

Mehmet Akdel, Douglas E.V. Pires, Eduard Porta-Pardo, Jurgen Janes, Arthur O Zalevsky, Balint Meszaros, Patrick Bryant, Lydia L. Good, Roman A. Laskowski, Gabriele Pozzati, Aditi Shenoy, Wensi Zhu, Petras Kundrotas, Victoria Ruiz-Serra, Carlos H.M. Rodrigues, Alistair S Dunham, David F Burke, Neera Borkakoti, Sameer Velankar, Adam Frost, Kresten Lindorff-Larsen, Alfonso Valencia, Sergey Ovchinnikov, Janani Durairaj, David B Ascher, Janet M. Thornton, Norman E Davey, Amelie Stein, Arne Elofsson, Tristan Croll, Pedro Beltrao

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods have led to protein structure predictions that have reached the accuracy of experimentally determined models. While this has been independently verified, the implementation of these methods across structural biology applications remains to be tested. Here, we evaluate the use of AlphaFold 2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modelling of interactions; and modelling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modelled when compared to homology modelling, identifying structural features rarely seen in the PDB. AF2-based predictions of protein disorder and protein complexes surpass state-of-the-art tools and AF2 models can be used across diverse applications equally well compared to experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life science research.

9: Recording of "sonic attacks" on U.S. diplomats in Cuba spectrally matches the echoing call of a Caribbean cricket
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Posted 04 Jan 2019

Recording of "sonic attacks" on U.S. diplomats in Cuba spectrally matches the echoing call of a Caribbean cricket
10,883 downloads bioRxiv biophysics

Alexander L. Stubbs, Fernando Montealegre-Z

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.

10: RELION-3: new tools for automated high-resolution cryo-EM structure determination
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Posted 19 Sep 2018

RELION-3: new tools for automated high-resolution cryo-EM structure determination
10,325 downloads bioRxiv biophysics

Jasenko Zivanov, Takanori Nakane, Bjoern Forsberg, Dari Kimanius, Wim JH Hagen, Erik Lindahl, Sjors HW Scheres

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.

11: Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant
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Posted 13 Jan 2021

Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant
9,774 downloads bioRxiv biophysics

Gard Nelson, Oleksandr Buzko, Patricia R Spilman, Kayvan Niazi, Shahrooz Rabizadeh, Patrick Soon-Shiong

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.

12: Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision
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Posted 05 Dec 2018

Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision
9,755 downloads bioRxiv biophysics

Simao Coelho, Jongho Baek, Matthew S Graus, James M Halstead, Philip Nicovich, Kristen Feher, Hetvi Gandhi, Katharina Gaus

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.

13: HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.
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Posted 21 Feb 2019

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.
9,223 downloads bioRxiv biophysics

C. Favard, J. Chojnacki, P. Merida, N. Yandrapalli, J. Mak, C. Eggeling, D. Muriaux

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.

14: Real-time cryo-EM data pre-processing with Warp
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Posted 14 Jun 2018

Real-time cryo-EM data pre-processing with Warp
8,440 downloads bioRxiv biophysics

Dimitry Tegunov, Patrick Cramer

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.

15: Theoretical basis for stabilizing messenger RNA through secondary structure design
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Posted 24 Aug 2020

Theoretical basis for stabilizing messenger RNA through secondary structure design
8,329 downloads bioRxiv biophysics

Hannah K Wayment-Steele, Do Soon Kim, Christian A. Choe, John J. Nicol, Roger Wellington-Oguri, R. Andres Parra Sparberg, Po-Ssu Huang, Eterna Massive Open Laboratory, Rhiju Das

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.

16: Expansion microscopy at one nanometer resolution
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Posted 05 Aug 2022

Expansion microscopy at one nanometer resolution
8,149 downloads bioRxiv biophysics

Ali H. Shaib, Abed Alrahman Chouaib, Vanessa Imani, Rajdeep Chowdhury, Svilen Veselinov Georgiev, Nikolaos Mougios, Mehar Monga, Sofiia Reshetniak, Daniel Mihaylov, Han Chen, Parisa Fatehbasharzad, Dagmar Crzan, Kim Ann Saal, Claudia Trenkwalder, Brit Mollenhauer, Tiago F. Outeiro, Julia Preobraschenski, Ute Becherer, Tobias Moser, Edward S Boyden, A. Radu A. Aricescu, Markus Sauer, Felipe Opazo, Silvio Rizzoli

Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

17: Structure of replicating SARS-CoV-2 polymerase
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Posted 27 Apr 2020

Structure of replicating SARS-CoV-2 polymerase
8,126 downloads bioRxiv biophysics

Hauke S. Hillen, Goran Kokic, Lucas Farnung, Christian Dienemann, Dimitry Tegunov, Patrick Cramer

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.

18: SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome
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Posted 30 Jun 2020

SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome
8,112 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.

19: RNA velocity unraveled
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Posted 13 Feb 2022

RNA velocity unraveled
7,734 downloads bioRxiv biophysics

Gennady Gorin, Meichen Fang, Tara Chari, Lior Pachter

We perform a thorough analysis of RNA velocity methods, with a view towards understanding the suitability of the various assumptions underlying popular implementations. In addition to providing a self-contained exposition of the underlying mathematics, we undertake simulations and perform controlled experiments on biological datasets to assess workflow sensitivity to parameter choices and underlying biology. Finally, we argue for a more rigorous approach to RNA velocity, and present a framework for Markovian analysis that points to directions for improvement and mitigation of current problems.

20: ProSPr: Democratized Implementation of Alphafold Protein Distance Prediction Network
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Posted 04 Nov 2019

ProSPr: Democratized Implementation of Alphafold Protein Distance Prediction Network
7,724 downloads bioRxiv biophysics

Wendy M Billings, Bryce Hedelius, Todd Millecam, David Wingate, Dennis Della Corte

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.

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