Oct 16, 2023
Sample vitrification and cryo-EM data acquisition
- 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Protocol Citation: Minghao Chen 2023. Sample vitrification and cryo-EM data acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39rreg25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82920
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000350
Abstract
Sample vitrification and cryo-EM data acquisition
Sample vitrification
Sample vitrification
Glow-discharge the grids at 25 mA for 30 sec with PELCO easiGlow system (Ted Pella)
Apply 3 μl of protein solution to the grid:
QUANTIFOIL R1.2/1.3 mesh 300 (Electron Microscopy Sciences), or
QUANTIFOIL R2/1 mesh 300 (Electron Microscopy Sciences)
Vitrify the sample with a Vitrobot cryo-plunger (Thermo Fisher Scientific)
(Optional) Add 0.05%(w/v) of n-Octyl-Beta-D- Glucopyranoside as a surfactant before vitrification.
cryo-EM data acquisition at 300 kV Titan Krios microscope
cryo-EM data acquisition at 300 kV Titan Krios microscope
Collect dataset at a magnification of 81,000x and a corresponding pixel size of 1.05 Å and a defocus range of -0.8 to -2.0 μm. Image stacks contain 50 frames with a total dose of 50 e/Å2.
cryo-EM data acquisition at 200 kV Talos Arctica microscope
cryo-EM data acquisition at 200 kV Talos Arctica microscope
Collect dataset at a magnification of 36,000x and a corresponding pixel size of 0.5575 Å and a defocus range of -0.8 to -2.0 μm in a super-resolution correlated-double sampling mode. Image stacks contain 50 frames with a total dose of 50 e/Å2.