About NICE-NIH Intramural CryoEM Consortium 

NIH Intramural CryoEM Consortium (NICE) serves intramural investigators in all NIH IC’s. NICE provides access to state-of-the-art Titan Krios cryo-electron microscopes for atomic-resolution structure determination of protein, macromolecular complexes, membrane receptors, cellular organelles, and infectious pathogens, using single particle or cryo-tomography approaches. Fine detailed 3D maps computed by electron micrographs spark numerous biological insights in cancer research, infectious disease research, vaccine design, cell development, and human physiological and functional studies.

Applications:

The following are used for microscopy imaging.

• Negative Staining and cryoEM sample preparation.
• Negative Staining and cryoEM sample screening.
• CryoEM Single Particle and Tomography data collection

Instruments:

Building 37

• Titan Krios 300kV Electron Microscope with Gatan BioQuantum Image Filter and K3 Direct Electron Detector
• FEI Vitrobot Mark4
• Leica Cryo GP2
• Ted Pella Easy Glow grid discharging system

Building 33

• Glacios 200kV Electron Microscope with Selectris Image Filter and Falcon 4i Direct Electron Detector
• Contact: Dr. Haotian Lei haotian.lei@nih.gov

Building 50

• Tecnai 12 120kV Electron Microscope with K2 summit
• 2 Gatan 626 cryo holders
• 1 multi-specimen cryo holder
• Gatan turbo pumping stations for cryo holders

NICE Advisory Committee

Director: Joseph Macrotrigiano Ph.D. (NIAID)

Deputy Director: Di Xia Ph.D. (NCI)

Deputy Director: Jenny Hinshaw Ph.D. (NIDDK)

Operational Procedure and Policy for the NCI Krios Affiliated with NIH IRP CryoEM Facility (NICE) Rick Huang, Ph.D. Reviewed by the NICE Advisory Committee 2-28-2023

1. Goals:

• To serve structural biologists of the NIH intramural program
• To provide access to high-resolution EM data collection on the Titan Krios, not for screening
• To provide access to initial sample screening on the T12.

2. Sessions:

• Krios scope time is divided into sessions. A typical session will be 3 days: Monday-Wednesday and Thursday-Saturday.
• Krios scope will perform cryo cycle on every Sunday late afternoon no later than 5pm to remove contamination from the scope.

3. Data collection:

• One dataset per session is strongly encouraged for best efficiency and result, unless for multi-conformational samples or pushing high resolution.
• 2-3 overnight datasets (same imaging condition but in different protein complexes, states, or drug binding substrate) can be collected in one session. Users need to include this request in the proposal or notify the facility staff at least 2 weeks in advance.
• If necessary, one session in each month is reserved for the facility internal testing, scheduled maintenance, microscope alignment or service when needed.
• Users must collect with the sample stated in their proposal. Any change to a non-approved project is prohibited and may result in cancellation.
• In the unlikely case of “unpromising” sample (poor protein quality), as agreed by the user, data collection will be terminated.

4. Special requests:

• Our microscope is currently optimized for dose-symmetric tomography, SPA standard collection, multi-shots with image shift, and tilted SPA (up to +45deg). When data collection requires new methods or functions, please include the request in the proposal or inform the facility staff as least 4 weeks in advance. For example, installation new version of serialEM or a new data collection method that is not yet available.

5. Error reporting:

• Hardware or software error: When encountering any hardware or software error, ALL USERS must close the “column valve” and report the incident immediately. Please do not try to attempt to clear any major error message because the facility staff will need to record and report the specific error to the service engineer for troubleshooting.
• For minor error messages, advanced users shall record a screenshot and document the date and time of the incident.

6. Grids loading and screening:

• Grid clipping and loading
  • Users are encouraged to clip their grids in advance.
  • Facility staff or advanced users can clip the grids on the day of the session (Monday or Thursday).
  • Facility staff can only clip ~4 grids (up to 8 if necessary) for the 3-day session.
  • Up to 11 pre-clipped grids can be loaded into the autoloader.
  • Reloading cassette during the session is prohibited. Multiple loading within a day will result autoloader jam and malfunction.
  • Only experienced advanced users are allowed to load grids into the cassette.

• Grid screening is IMPORTANT
  • Heavy grid screening, e.g. checking all 12 grids in the autoloader, which may result in cancellation if none of the grid looks promising.
  • Screening is expected to complete in the morning on day 1. Users continuing to screen grids in the afternoon is an indication that this sample has not yet been checked on the screening microscope.
  • All users should prioritize the grids selection. Systematic/planned screening can be done quickly to ensure data quality, e.g. grids with different conditions.

7. User types:

• All users
  • Loading or changing grids at nights, weekends, or holidays is never allowed. If the project is “mission critical”, night, weekend, holiday work must be approved by the facility manager. Staff support and on-site engineers will not be available for troubleshooting outside of NIH business hour.
  • All users can add more targets using remote desktop app anytime.
• Beginner
  • Beginners are defined as first time or infrequent users. Data collection will be fully assisted by facility staff. The facility staff will show the users procedures and provide necessary training. Users can only pick targets for data collection.
  • A facility staff must be in the control room with beginner users during the session.
• Intermediate
  • Intermediates are defined as experienced, returning, and frequent users, e.g. at least once a month on the Krios and once a week on the screening microscopes.
  • Intermediate users can take control of setting up data collection. Facility staff will perform microscope alignments and provide assistance when needed.
  • At least one facility staff must be available on campus when intermediate users set up data collection in case of hardware malfunction.

• Advanced
  • Advanced are defined as frequent users who collected data on the NCI-building 37 Krios for many years and have full understanding of the microscope and the camera system.
  • Advanced users can take full control of setting up data collection and normal microscope alignments (direct alignment only).
  • If any high-level scope/camera setting needs to be changed, please confirm with facility staff for approval.
  • Advanced users must return scope/camera settings to the original settings at the end of the session.

8. Session schedule:

• Time is awarded to users as unit of sessions. No more than two sessions shall be awarded to a user at one time.
• Microscope session will be scheduled 1-2 months in advanced. Users must submit a proposal to request for every scope session. Proposals are reviewed by the NICE advisory committee.
• Schedule calendar will be available for users to access (viewing only).
• NCI and some advance users can request to be on “standby list”. If there is any sudden cancelation, the time will be granted to the user on the standby list, who should have the sample prepared and ready to load when time becomes available.
• If the sample is determined to be “unpromising”, then the facility has the right to cancel the session and then give the remaining time to the users on the standby list. For example, ice extremely thin or thick, no particles, particles with wrong dimensions, crystal ice …etc.
• Due to unexpected hardware failure leading to cancelling session, the session will be rescheduled to the earliest time in the next cycle or be placed at the top of the standby list in order to minimize waiting time.

9. Publication and authorship:

• Acknowledgement in manuscripts
  • Acknowledgement in the manuscript is required if the data is collected on the NCI-Bdg37 Krios.
    • Example:
      • We would like to thank Dr. Rick Huang for help with cryoEM data collection. This work utilized the NCI/NICE Cryo-EM Facility.  This work utilized the NCI/NICE Cryo-EM Facility.
      From 2020-08-01 to 2022-06-01
      • We would like to thank Ms Allison Zeher and Dr. Rick Huang for help with cryoEM data collection. This work utilized the NCI/NICE Cryo-EM Facility.
      From 2022-06-01 to 2024-06-01
      • We would like to thank Ms Zabrina Lang, Mr. Abraham J Morton, and Dr. Rick Huang for help with cryoEM data collection. This work utilized the NCI/NICE Cryo-EM Facility.
• Collaboration with co-authorship
  • Authorship should be granted to the facility staff who contributes in addition to data collection.
  • Data collection requires special customization or testing new methods.
  • Assisting and consulting users on data processing.
  • Providing helpful suggestions on sample preparation, troubleshooting, and sample optimization.

The following files are templates for proposal submission for:

Krios

NICE Krios Proposal Template 2023

Glacios

NICE Glacios Proposal Template 2023