Single Particle Analysis with Cryo EM

Single Particle Analysis for purified protein complexes.

Workflow:

1. Wash holey carbon membrane/ gold grids with pure acetone (as required)
2. Natural dry
3. Glow discharge the grid by the Quorum GloQube
4. Prepare graphene-oxide membrane on the gold grid (as required)

• Graphene oxide dispersion in H2O (Sigma-Aldrich, Merck)
• Quantifoil, C-flat, UltraAuFoil
• Anti-capillary crossover tweezers

5. Prepare ultra-thin carbon membrane on the gold grid (as required)

• Carbon coats on mica
• Quantifoil, C-flat, UltraAuFoil

6. Put the purified protein-complex on the designed grid as above
7. Blot the access volume on the grid with filter papers
8. Vitrification purified protein-complexes on the grid by the FEI Vitrobot
9. Set the frozen grid on the Gatan cryo-transfer holder
10. Insert the cryo-holder to the FEI TalosF200C
11. General alignment for low electron-dose imaging
12. EPU image acquisition after the Atlas
13. Image processing by the Relion or CryoSparc on the Cryo-EM GPU server
14. Repeat several refinements
15. 3D-model