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Protocol publication

[…] For sample preparation, 200 mesh Quantifoil R2/2 copper grids were glow-discharged and 0.65 mg/mL of the CCV preparation in 100 mM MES, 1 mM EGTA, 0.5 mM MgCl2 was added to the grid. The sample was plunge frozen with an FEI Vitrobot Mark V in liquid ethane.The data were collected on a Talos Arctica (FEI) at 200 kV with a field emission gun and a nominal magnification of 45,000 at the Cryo-EM Swedish National Facility. The images were collected with a Falcon II and a dose rate of 9.53 e- per Å2 per second. The recording time was 2.5 s, resulting in 30 frames and a total dose of 23.8 e- per Å2. The frames were motion corrected and dose weighted with MotionCorr () and the CTF was estimated with Gctf (). The pixel size for the images was 3.25 Å/pixel. For the processing, images with a box size of 130Åx130Å were manually extracted with RELION (). A total of 43,711 manually-picked individual particles from 2836 images were used for the 2D classification through RELION 2.0. After the classification, a new stack was formed for one vesicle based on the vesicle size. The image stack-classified into 16 classes with no (C1) symmetry imposed. The particles from the most populated well-defined 3D class were combined into a final stack. This stack was re-classified in three classes with D6 symmetry enforced. The particles of the most populated class were refined within RELION in D6. The gold standard FSC was calculated in RELION. Molecular graphics and analyses were performed with the UCSF Chimera package, a visualization system for exploratory research and analysis, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) (). The structure has been deposited with the EMDB-ID #4335. […]

Pipeline specifications

Software tools MotionCorr, Gctf, RELION, UCSF Chimera
Application cryo-EM
Organisms Mus musculus
Chemicals Adenosine, Adenosine Triphosphate