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 14.3 10.6019/EMPIAR-10321 EMDB EMD-10309 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 with high intracellular calcium (scs2/22 ist2 deletion and GCaMP expression) /data/Tiltseries/TS_frames_aligned_reordered tilt series MRC MRC 121 4 1 4 SIGNED 16 BIT INTEGER 3710 3.753 3838 3.753

The cells were treated with 200 mM calcium chloride directly before plunge freezing. Cells with high GCaMP fluorescence by cryo-FM were targeted for 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. Tilt-series were acquired between +/- 60 degrees starting from 0 degrees with 1 degrees 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 -5 micron defocus. A dose of 1.0 e-/square angstroms was applied per image of the tilt-series. The tilt-series frames were aligned and then reordered by tilt angle using IMOD.