REL 2019-09-23 2019-11-18 2019-11-18 0000-0002-2778-3936 Wanda Kukulski Cell Biology Division, MRC Laboratory of Molecular Biology Francis Crick Ave Cambridge Cambridgeshire United Kingdom CB2 0QH 0000-0002-2778-3936 Wanda Kukulski Cell Biology Division, MRC Laboratory of Molecular Biology Francis Crick Ave Cambridge Cambridgeshire United Kingdom CB2 0QH Hoffmann PC Bharat TAM Wozny MR Boulanger J Miller EA Kukulski W 3.1 10.6019/EMPIAR-10322 EMDB EMD-10310 Hoffmann PC Bharat TAM Wozny MR Boulanger J Miller EA Kukulski W Developmental Cell Dev Cell 4 51 2019 10.1016/j.devcel.2019.09.019 cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overexpressed and scs2/22 ist2 tcb1/2 are deleted /data/Tiltseries tilt series MRC MRC 117 1 SIGNED 16 BIT INTEGER 3710 3.753 3838 3.753

Overexpression of Tcb3-GFP in the cells was induced by addition of 1% galactose to the medium. Cells were then plunge-frozen and subjected to cryo-FIB milling. Electron cryo-tomographic tilt-series were collected on a Titan Krios (FEI) operated at 300 kV using a Quantum energy filter (slit width 20 eV) and a K2 direct electron detector (Gatan) in counting mode at a pixel size of 3.7 angstroms and at a dose rate of ~ 2-4 e-/pixel/second on the detector. The tilt-series were acquired between +60 and -56 degree starting from 0 degree with 1 degree increment using SerialEM (Mastronarde, 2005) following a grouped dose-symmetric acquisition with a group size of 4 (Bharat et al., 2018; Hagen et al., 2017), and at -4.5 micron defocus. A dose of 1.2 e-/square angstroms was applied per image of the tilt-series. The tilt-series images were reordered by tilt angle.