Sep 30, 2023

Public workspaceCryo-EM Grid preparation

This protocol is a draft, published without a DOI.
  • 1University of California, Berkeley;
  • 2Aligning Science Across Parkinson's
Annan SI Cook
Open access
Protocol Citation: Annan SI Cook 2023. Cryo-EM Grid preparation. protocols.io https://protocols.io/view/cryo-em-grid-preparation-c2pbydin
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: September 22, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88515
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol details Cryo-EM grid preparation.
Attachments
852-2198.pdf
852-2198.pdf
36KB
Materials
Materials

  • PI3KC3-C1~RAB1A sample with n-Octyl-β-D-Glucopyranoside (OG) at a final concentration of 0.05%
  • QUANTIFOIL R 2/1 mesh Cu 300 holey carbon grids
  • Vitrobot cryo-plunger (Thermo Fisher Scientific)
  • Cryo-EM microscope (300 kV Titan Krios with X-FEG and energy filter)
  • K3 Summit direct electron detector (Gatan)
  • SerialEM software
  • Liquid nitrogen
  • Humidity control system
Grid Preparation
Grid Preparation
1m
Freshly glow-discharge the QUANTIFOIL R 2/1 mesh Cu 300 holey carbon grids using a glow-discharge system (PELCO easiGlow) at 25 mA current for Duration00:01:00 .
1m
Sample Application
Sample Application
Mix the PI3KC3-C1~RAB1A sample with 0.05% n-Octyl-β-D-Glucopyranoside (OG) to achieve the desired final concentration.
Apply a small volume (typically Amount3-4 µL ) of the sample onto the glow-discharged side of the grids.
Vitrification
Vitrification
3s
Load the grids with the sample into the Vitrobot cryo-plunger.
Set the Vitrobot to the following conditions:
Maintain 100% humidity inside the chamber.
Set the temperature to Temperature4 °C .

Wait for Duration00:00:03 .

3s
Blot using a blotting force of -15 (may need calibration depending on your particular instrument) .
Data Acquisition Setup
Data Acquisition Setup
Prepare the Cryo-EM microscope (300 kV Titan Krios) for data acquisition.
Used X-FEG and an energy filter set to 20 eV.
Data Collection Parameters
Data Collection Parameters
Use SerialEM software for automated data collection.
Set the following parameters for data collection:

  • Magnification: 81,000x.
  • Super-resolution pixel size: 0.525 Å.
  • Defocus range: -0.8 to -2.2 micrometers.
  • Collect 50-frame image stacks.
Data Collection
Data Collection
Initiate the data collection process using the specified parameters.
Monitor the data acquisition progress and ensure that the images are being recorded.
Cumulative Dose Control
Cumulative Dose Control
Each movie has a cumulative electron dose of 50 e/Ų.