EM Tips

General Advice

1. Do the tutorials with sample data! Take a look at the output and how long different calculations take

   1. Get sample data from software developers or from http://challenges.emdataresource.org/?q=map-challenge-targets

2. Refer back to the tutorials as you process your own data.

3. Search the mailing lists + http://lmgtfy.com/ before you ask questions. You'll learn a lot!

   1. Does an email to the mailing list feel too scary? Try tweeting about your question with #cryoEM. To me, twitter feels more casual, and you can always delete your tweet (though things never really leave the internet).

4. Have a question about a paper? Don't be afraid to reach out to the authors. I've found people are very willing to help you out if you ask a specific question. Scientists love to talk about their work! You'll probably have better luck emailing grad students and post docs rather than the PI since they don't get as many emails.

Sample preparation for Microcrystal Electron Diffraction (MicroED)

Mailing lists

• 3DEM - http://3dem.ucsd.edu/mailinglist.shtm

  • General EM posts

• CryoSPARC - https://discuss.cryosparc.com/

• EMAN2 - https://groups.google.com/forum/#!forum/eman2

• EMAN2 and Sparx for Developers - https://groups.google.com/forum/#!forum/eman2-developers

  • have an installation issue? Search there!

• FreAlign - http://grigoriefflab.janelia.org/forum

• Relion CCP-EM list https://www.jiscmail.ac.uk/cgi-bin/webadmin?A0=CCPEM

• Sphire - https://listserv.gwdg.de/mailman/listinfo/sphire

Treating electron microscopy grids with Graphene Oxide

• Adding graphene oxide to your grid is a great way to change the distribution of your protein of interest. Amorphous carbon sheets can be added to grids, but they create high background noise, so it's hard to see small proteins. Graphene oxide has a much lower contribution to background noise.

• Several protocols for making graphene oxide grids exist on the internet.

  • Video by Thomas Martin in the Scheres group at LMB

  • Instructions from Kangkang Song at UMass

  • Protocol for Graphene oxide flakes by Martin Cheung

  • Protocol by Eugene Palovcak in the Cheng lab

  • Purchase grids that are already treated with graphene oxide

• Sara's advice

  • Recently, I started sonicating my 0.2 mg/mL graphene oxide solution for 15 to 60 seconds before use. This has made a HUGE difference in the uniformity of my graphene oxide grids. I can’t emphasize enough how important this is!

  • I typically make my graphene oxide grids about 30 minutes before I use them to freeze protein. If I can’t use them right away, I store them under vacuum to minimize contamination from the air.

  • Our lab usually uses the EmiTech K100x at 20 mA for 60 s or the Pelco EasiGlow at 15 mA for 60 s.

    • I found these glow discharging conditions were insufficient for the LMB protocol. I got poor coverage of graphene oxide on my grids.

    • Now I use the EmiTech k100x 50 mA for 120 s for Quantifoil carbon/copper grids and Au Ultrafoils. I initially thought 50 mA would totally destroy the carbon on my grids, but I was pleasantly surprised to find it didn't. It just made the grids very hydrophilic so they took up the graphene oxide well using the LMB and UMass protocols.

    • I'm still optimizing because I often find a few pieces of graphene oxide over my holes.

  • For one of my membrane proteins, using graphene oxide reduced the number of particles I find in my GO-covered holes. Usually I think of substrates as a way to concentrate my sample, but I have the reverse with membrane proteins.