Cryo-EM Training

 

Basic EM training in the Purdue Electron Microscopy Facility with Dr. Gilpin:

  1. 1. Before requesting cryo-EM training, please complete basic EM training with Dr. Chris Gilpin (glipin@purdue.edu)in the Purdue Electron Microscopy Facility
  2. 2. Request training for the T20 and T12. Both microscopes will be useful for screening potential cryo-EM samples.


To arrange cryo-EM training at the Purdue Cryo-EM Facility (PCEMF):

  1. 1. Contact Dr. Kunpeng Li via e-mail (li2182@purdue.edu) requesting a training session between 1pm-8pm on Tues-Fri
  2. 2. Complete the Purdue University Laboratory Safety Fundamentals (LSF) online training:http://purdue.vividlms.com
  3. 3. Bring the printed certificate of completion to the first cryo-EM training session


Cost comparison between training and purchasing full service

There are two options when considering using the Purdue Cryo-EM facility: 1) personnel training or 2) purchasing full service. If you only have one specific project involving cryo-EM, it will be cheaper to purchase a full service session, which will yield results faster. If you anticipate your project will require more than a few sessions on the electron microscope it would be wise to receive full training and certification on the instruments. For personnel training, it is estimated ~12 hours of microscope time is needed at $98/hr. In total, the training sessions can be completed in one month with an additional 10 hours of practice sessions with your sample under supervision.

Cryo-EM Training on the CM200

To reserve time on theTitan, the PCEMF requests users provide example micrographs demonstrating good ice and visible particles. This is to ensure the sample quality is good enough to justify Titan usage. We do not guarantee optimized cryo-EM sample conditions after completing cryo-EM training, but our traning services will provide the skills needed for users to independently optimize their sample conditions for cryo-EM.

The CM200 training workflow:

cryoEM training schedule

After finishing the cryo-EM training, users still need 10 more supervised sessions on the microscope with their sample. At the conclusion of the training sessions, users will be certified once they have passed a final exam. The facility team will work with you until you get a satisfactory dataset. Below are relavent links for cryo-EM training:

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      1. PCEMF training plan and schedule: Purdue_cryoEM_training.pdf
      2. CM200 operation procedure:  CM200_manual
      3. Final exam: CM200_test

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Relevant cryo-EM tutorials:

1) Basic cryo-EM concepts and procedures (Vanessa Cabra, Montserrat Samsó)

2) Grid preparation to image acquisition (David S. Booth, Agustin Avila-Sakar in Yifan Cheng Group)

3) Glow discharging grids with the Emitech K950x  (Brenda Gonzalez in Wen Jiang Group):

4) Sample prep protocols optimized for small, asymmetric proteins (Ren Gang)

5) Making GO grids (Thomas G. Martin in Sjors H. W. Scheres Group)



other useful tutorials:

Scarff CA, Fuller MJG, Thompson RF, Iadaza MG. Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems. J Vis Exp 2018 Feb 6;(132). Available from: http://dx.doi.org/10.3791/57199 PMID: 29443097

 

Titan training (only for advanced users)

We do not have a specific training regimen for the Titan. However, if users have good enough sample for data collection on the Titan, the staff will guide users through the Titan operation and data collection process. For more experienced cryo-EM users, there are additional training options including instructions on:

      • Autogrid assembly for the Titan

      • Loading grids into cassettes for Titan usage

      • Installing autoloader cassettes into the Titan

      • Direct alignment, objective lens stigmator and comma free alignment of the Titan

      • K2 camera and DM software operation

      • Titan column/autoloader conditioning and K2 cryo-cycle

      • Leginon data collection and Appion data processing for Titan data collection

For new users wanting to learn about Titan operation, it is recommended to watch the staff or senior students operating the Titan during data collection. Learning how to operate the Titan is a lengthy process and typically requires many sessions to watch and learn.