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Center for Structural Biology

Training

Specimen Preparation Vitrobot Training Module 

Before Training Module 

  1. Watch Grant Jenson Lectures on cryo-specimen preparation.
  2. Read Vitrobot Protocol on CryoEM Core Website. 
  3. Have iLAB account Setup. 
  4. Contract the Core Director and schedule a training session. 

Training Module 

  1. Overview of Vitrobot and other equipment. 
  2. Overview of TEM grids, plasma cleaner and handling of TEM grids. Have the student pickup old grids with pair of forceps on slide or TEM grid block, and cryo-grid box. 
  3. Core Staff prepare cryo-grids for students while talking through process, student takes notes on handout (Vitrobot Protocol or Quick Start Guide). 
  4. Core Staff store cryo-grids in Dewar, explain process and inventory and the Vitrobot Notebook. 
  5. Students prepare cryo-grids (1) without LN2 or (2) with LN2 but using water and old TEM grids. Core Staff watch students and provide feedback or help. 

After Training Module 

  1. Students practice handling grids on their own time. 
  2. Students schedule assessment sessions with cryoEM Core Staff. Core staff watch students prepare their own cryo-grids with a sample or water. Core Staff use Rubic to score students, passing grade of 3 or higher is required. 
  3. If a student passes, they can book and operate the Vitrobot without Core Staff.  
  4. If a student fails, they can do an assisted Vitrobot freezing experiment with Core Staff and take assessment again. 

 

Talos Arctica Training Module

Before Training Module 

  1. Watch Grant Jenson Lectures on cryoTEM https://cryo-em-course.caltech.edu/unit-1-outline
  2. Read CryoEM 101 Chapter 3 Grid Screening & Evaluation and Chapter 4 Cryo-EM Data Collection (however, all chapters are recommended). https://cryoem101.org/
  3. Visit the cryoEM Core website to learn about the policies, rigor reproducibility, and protocols related to cryoEM. Read the operation of the Talos Arctica protocol, SerialEM Lecture, and watch videos on operation of the microscope. 
  4. Set up an iLAB account. https://uncch.ilab.agilent.com/account/login
  5. Setup Globus Account. https://www.globus.org
  6. We recommend that the student watches a person operate the microscope (ideally from their group).

Training Module 

Goals: Learn about the microscope configuration, camera settings and how to use SerialEM to operate the microscope.

  1. Core Staff operates the microscope for the student while explaining the process. During this process the student will take notes on the Talos Arctica protocol handout from the core website and is expected to ask questions.If the student does not have their cryo-grids, the core can provide the student with cryo-grids of mouse apoferritin or something similar.

Topics covered during the training session include:

    1. Alignment of the TEM.
    2. Using the Autoloader.
    3. Low-dose imaging.
    4. Assessing sample quality and ice thickness.
    5. Acquiring and saving images.
    6. Collecting montages.
    7. Data collection using the serialEM GUI and scripts.
    8. Optional topics will be covered depending on the goals of the student and what imaging modality they are using, ie. single-particle cryoEM, tomography, microED, cryoTEM.
  1. The student will operate the microscope under the supervision of the core staff, either during the same training session or during a separate training session at a later date.

Optional advanced training If the Student is collecting data (single-particle, tomography, microED) overnight:

    1. Direct alignment of the TEM.
    2. Power-cycle the Gatan K3 camera, and restart the microscope computer.
    3. Pre-processing data with cryoSPARC Live.
    4. Setting up low-dose settings in SerialEM.

Optional Training Module 

  1. Direct alignment of the TEM.
  2. Power-cycle the Gatan K3 camera, and restart the microscope computer.
  3. Pre-processing data with cryoSPARC Live.
  4. Setting up low-dose settings in SerialEM.

After Training Module

  1. Student reviews their notes taken during a training session on their own.
  2. Students scheduled an assessment session with the cryoEM Core Staff. Core Staff watch students operate the microscope. Core staff use a Rubric to score the students, passing grade of 3 or higher is required.
  3. If the student passes they can operate the microscope independently.
  4. If the student has deficiencies or is uncomfortable operating the microscope independently further training sessions with the core staff and reassessment is possible.

List of SerialEM Settings and Scripts

Single Particle Settings File “20231014_SP_45K” 

  1. SingleMontage – collect full grid montage of grid.
  2. Map2grids – collects full grid montages of multiple cartridges.
  3. SquareCenteringTiltV2 – centers stage over grid square – useful when collecting medium mag montages.
  4. Multishot_Filter_threshold – skips acquire points with less than set threshold of shots per stage shift. Used in tandem with data collection scripts that use BIS method.
  5. DoseCal – calculates total dose and prints imaging conditions for record.
  6. UNCChainSawV2– Collects one multishot in center of grid square. Our quick and fast SP data collection script.  
  7. Nothing2Fancy – Script will collect search, view and record and save as mrc and jpg for points added.
  8. NothingFancy – Script will collect view and record as mrc and jpg for points added.
  9. HoleCenterLoop – test hole center.
  10. Tilt_Check – checks if you can tilt the stage at the view level.
  11. MMM_HoleFinder – finds holes in medium mag montages. Run this script before data collection script for SP BIS.
  12. DataCollection_V6 – Most current SP BIS data collection script.
  13. HoleHunter – finds holes in image, useful for setting up multishot for BIS.

Anything above 14 is for testing  

Youtube Channel

The UNC-CH CryoEM Core YouTube Channel is committed to providing educational content related to biological cryo-electron microscopy (cryoEM), encompassing specimen preparation and the operation of the electron microscope. We post content on cryoTEM of nanoparticles, SP cryoEM of macromolecular complexes, CryoET, and microED. The content is mainly for students, post-doctoral fellows, and researchers utilizing our core facility. Our goal is to provide information to individuals interested in learning the technical aspects of cryoEM. We recognize that each laboratory, core, or center may have distinct policies, procedures, and equipment. Therefore, viewers are urged to consider these variations when viewing the content on this channel. We strive to provide accurate and reliable information, but we cannot guarantee the completeness of the content.