ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines. Hoffmann et al
Supplemental Video 1 from the manuscript "ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines" by Hoffmann et al. Tomographic reconstruction of purified S-EABR eVLPs, related to Figure 1. Representative eVLPs are highlighted in boxes.
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Cryo-ET of S-EABR eVLPs SEC-purified S-EABR eVLPs were prepared on grids for cryo-ET using a Mark IV Vitrobot (Thermo Fisher Scientific) operated at 21°C and 100% humidity. 2.5 μL of sample was mixed with 0.4 μL of 10 nm fiducial gold beads (Sigma-Aldrich) and applied to 300-mesh Quantifoil R2/2 grids (GOQ300R22Cu10; Quantifoil Micro Tools), blotted for 3.5 s, and then plunge-frozen in liquid ethane cooled by liquid nitrogen. Image collections were performed on a 300 kV Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at a nominal 42,000x magnification. Tilt series were collected on a K3 direct electron detector (Gatan) with a pixel size of 2.15 Å•pixel-1 using SerialEM software.62 The defocus range was set to -5 to -8 μm and a total of 120 e- • Å-2 per tilt series. Images were collected using a dose-symmetric scheme63 ranging from -60° to 60° with 3° intervals. Tomograms were aligned and reconstructed using IMOD.64 To build a model of an S-EABR eVLP, coordinates of a SARS-CoV-2 S trimer (PDB 6VXX) were fit into spike densities in the reconstructed tomograms using ChimeraX.65,66 Positions and orientations of the S protein were adjusted in a hemisphere of the eVLP in which the spike density was of higher quality. A 55 nm sphere was adapted from a cellPACK model (cellPACK ID: HIV-1_0.1.6_6)67,68 and added to the model to represent the eVLP membrane surface.