Biology subjects

Bezault, A.

Publications and source records attributed to Bezault, A..

3 recordsLinked to original sources

bioRxiv2022

MEPSi: A tool for simulating tomograms of membrane-embedded proteins

The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question. Tools for a-priory assessment of the feasibility of samples to answer biological questions and how to optimize imaging parameters to do so would be a major advantage. Here we describe MEPSi (Membrane Embedded Protein Simulator), a simulation tool aimed at rapid and convenient evaluation and optimization of cryo-ET data acquisition parameters for studies of transmembrane proteins in their native environment. We demonstrate the utility of MEPSi by showing how to detangle the influence of different data collection parameters and different orientations in respect to tilt axis and electron beam for two examples: (1) simulated plasma membranes with embedded single-pass transmembrane IIb{beta}3 integrin receptors and (2) simulated virus membranes with embedded SARS-CoV-2 spike proteins. HIGHLIGHTSO_LITool to simulate tomograms of membrane-embedded proteins C_LIO_LIDetangles influence of data acquisition parameters from sample quality issues C_LIO_LIRapid evaluation and optimization of cryo-ET data acquisition parameters C_LIO_LIProof-of-concept provided with integrins and SARS-CoV-2 spike simulations C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/501771v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@120b44eorg.highwire.dtl.DTLVardef@1d9056org.highwire.dtl.DTLVardef@ef327aorg.highwire.dtl.DTLVardef@1daf773_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Characterization of heterogeneity in nanodisc samples using Feret signatures

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/501900v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19ea37aorg.highwire.dtl.DTLVardef@1fed1aeorg.highwire.dtl.DTLVardef@781962org.highwire.dtl.DTLVardef@ca7859_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LINew methodology to characterize nanodiscs based on Feret signatures C_LIO_LIFeret signatures distinguish nanodisc morphologies and compositions C_LIO_LIAnalysis is highly sensitive to sample quality C_LIO_LIMethod selected condition to solve structure of small membrane protein Bcl-xL C_LI

molecular biology↗

bioRxiv2022

ACE2 nanoparticles prevent cell entry of SARS-CoV-2

The continual evolution of SARS-CoV-2 has challenged the efficacy of many COVID19 vaccines and treatment options. One strategy that evades viral escape is using the entry receptor, human Angiotensin-Converting Enzyme 2 (hACE2). Soluble hACE2 receptor domains show potential as decoys but genetic modifications are necessary to provide sufficient efficacy. However, these engineered constructs are potentially susceptible to viral escape. We combined native hACE2 with viral vectors to form nanoparticles presenting hACE2 analogous to human cells. Cell-based viral infection assays and cryogenic in-situ tomography show that hACE2 nanoparticles sequester viruses through aggregation, efficiently blocking entry of SARS-CoV-2 and its variants in model cell systems and human respiratory tract explants using native hACE2. Thus, we show that hACE2 nanoparticles have high potential as pan-variant COVID19 therapeutics.

cell biology↗