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Center for Structural Biology

Guidlines for CryoEM

We follow specific operating procedures to ensure Rigor and Reproducibility for CryoEM.  We recommend researchers using the CryoEM Core follow these guidelines for their own projects.

Specimen Preparation

Preparing cryo-grids with the Vitrobot

  • Only trained users are permitted to operate the Vitrobot.  If you do not know how to operate the Vitrobot contact the cryoEM Core and request training. If you know how to operate the Vitrobot but are not comfortable using it please contact the CryoEM Core for assistance or a refresher. If you would like the CryoEM Core to help prepare cryo-grids for you please contact us.
  • When operating the Vitrobot please use the CryoEM Core’s SOP.
  • Fill-out the Vitrobot Log Notebook each time you prepare cryo-grids. We use this information for billing and to keep track of the Vitrobot performance.
  • Setup the Vitrobot and cycle through the operating procedure to make sure that it is working before preparing cryogrids with your sample. Make sure that the humidifier and temperature are stable.
  • Take one TEM grid (old ones are available at the core for you to use), pick it up with the Vitrobot forceps, and use it to test the Vitrobot blotting mechanism. Make sure that the blotting pads make contact with the TEM grid, and are working properly before preparing cryo-grids.
  • Take good notes when preparing cryo-grids. When operating the Vitrobot and PLECO easeiGlow record what setting you use to prepare each of your cryo-grids. Write down which TEM grids you used, Quantifoil and C-FLAT TEM grids will have the catalog and batch number written on the box.
  • When preparing cryo-grids to look carefully at the TEM grid as you apply the sample. If the liquid does not evenly spread over the TEM grid it is likely hydrophobic and will not form thin vitreous ice.
  • Examine your TEM grids with the Light Microscope before preparing cryogrids. Make sure the support film is intact, before and after plasma cleaning.
  • Clearly label your cryogrid boxes with a unique identifier. We recommend using “Initials” followed by ”Box Number” followed by the “date”. The cryo-grid box will have 4 slots.   Keep track of how the grids are placed in the grid box, we always use counter-clockwise convention.
  • If you are storing your grids in a Falcon tube, make sure it is clearly labeled (name, date, and box label).
  • If you notice an error when operating the Vitrobot contact the CryoEM Core immediately. If you encounter a mechanical error turn off the Vitrobot.  Do not attempt to fix the problem, recorded the error, if possible take a picture with your phone.

Cryo-electron microscopy

Operation of the microscope

  • Only trained users are permitted to operate the Talos Arctica.  If you would like to learn how to operate the microscope or need assistance please contact us we are happy to help.
  • Direct alignments are preformed daily, before collecting data using a grating replica TEM grid (2,160 lines/mm waffle pattern). This grid is stored in the Cassette in position #12, and available to use at all times.
  • A gain and dark references are collected before collecting data with the Ceta CCD and Gatan K3 direct electron detector.
  • To assess the performance of the microscope we collect at data set (~100-200) of Apoferritin and process the data with CryosSPARC or Relion. This is done routinely and after major changes or repairs to the microscope. Horse spleen apoferritin we typically will get 2.7 Å resolution based on FSC=0.143 gold standard, and 2 Å for mouse apoferritin.
  • The autoloader cassette can accommodate up to 12 cryo-grids. We do not load samples from multiple groups during a single imaging session to reduce the chances of mislabeling samples.   We keep a written recorded of which samples were loaded into the microscope autoloader.
  • Only a CryoEM Core Staff is permitted to load cartridges (cryo-grids) into the cassette and transfer the cassette into the microscope autoloader.
  • The Talos Artica is used for routine screening and data collection, each are important, and we describe best practices for both (see below).

Screening Cryo-Grids

  • When screening cryo-grids keep track of which grids are imaged, what regions of the grids are imaged, and what setting you used to acquire the images. We recommend using the SerialEM Navigator file and annotate the Navigator Items during screening, and saving the Navigator file periodically.  The Navigator file can be saved and re-loading with SerialEM and used as a record of the microscope session.  We also recommend taking a screenshot of the Full Montage after data collection to keep a record of which areas were imaged.  It is possible to image a cryogrid multiple times, but you can’t collect data over the exact same area multiple times due to radiation damage to the sample. We recommend saving the SerialEM log file, it contains information from the session that can be useful for troubleshooting.
  • For each of the cryo-grids collect a SerialEM Full Montage. This is the best way to determine how well the specimen preparation protocol (ie. Vitrobot parameters) worked. The Full Montages are used as a “fingerprint” of each cryo-grid, as each is unique, and can be used to verify the identity of a cryo-grid in the event that the samples are lost or misidentified.  Full Montages are also used to estimate how many movies can be collected on a cryo-grid.  This information is useful when planning overnight and extended data collection.
  • When screening cryo-grids make sure to acquire images at different magnifications.  We typically will take low-dose images with the Gatan K3 direct electron detector at 62X (for Full Montages), 210X (to see the grid squares and ice), 5,300 X (to see the ice and particles), and 45,000 X (to visualize particle morphology).
  • Keep track of the microscope settings you use to acquire images. To include magnification, pixel size, spot size, what apertures are inserted, exposure time, total dose, what camera you use to acquire the images, defocus, and how you acquire the images.  Some of this information will be included in the MRC header, so if you save the images as jpg (which most people do) you will not have that information in the files.
  • Sometimes the complexes will dissociate, or fall apart at the air-water interface, or in solution. Measure particle size (diameter) to estimate the mass of the complex in ice.  The diameter of the particles and the expected molecular weight of the complex should agree with one another.

Data Collection

  • Before collecting a large data set (> 100 movies), acquire a small data set, and pre-process the movies using CryoSPARC Live or Scipion. If the maximum resolution of the micrographs is > 5 Å then do not collect data on this grid (or area of the grid).  Move to another area of the grid, or exchange cryo-grids.  Typically, we obtain between 3 – 4 Å maximum resolution for data collected at 45,000 X (pixel size 0.91 Å) with the Gatan K3 direct electron detector.
  • We recommend collecting a small data set (100-200 movies), process the data, and obtain 2D class averages with the visible secondary structure before collecting a large data set. The 2D Class averages will be a good indicator of the feasibility of obtaining a high-resolution 3D structure.
  • Meta data is stored in the mdoc files (including pixel size and image shifts) for each of the movies.
  • Save movies as uncorrected Tiff files. A single gain reference (.mrc format) file will be generated in the working directory, you will use this file to run gain reference correction on the data during image processing (ie. Relion, CryoSPARCM, CisTEM ect..).