A standard setting for one of our Gatan GIF/K3 systems would be:
Titan Krios GIF/K3 configuration:
Exposure Settings: 1.6-2.0 s, 40 ms frames (40-50 frames)
e Flux: ~20 e-/px/s with ~45-58 e-/Å total dose
Pixel Size: ~0.83Å/px (TIFF LZW format in counting or bin2 with an image dimension of 5760×4092 px)
Energy Filter: 20 eV slit
Defocus Range: -0.6 µm to -2 µm (nominal)

If you would like modifications let the NCCAT User Office know ahead of your session.

The main way to monitor your data collection is through the image viewer through our web interface.

Please remember to use your NCCAT proposal session assignment email as your main correspondence thread including the NCCAT User Office so we can promptly see to your queries. As part of this process, it is critical that you include your NCCAT proposal ID in the subject of all emails. If you would like to set up a video conference to better strategize on a data collection strategy let the NCCAT User Office know.

Note that NCCAT’s business hours are 9am-5pm ET during business days. Email correspondences outside this time may be delayed.

Reports

On the upper menu in the webpage you may also access a “Report” page to give you a summary of the experiment. This report updates in real-time.

Please download your data via Globus. It may be easier to download the raw TIFF LZW or MRC data after a session is finished, but the Leginon aligned sum images (*enn-a-DW.mrc) are relatively small and that would be useful if you wanted to use them for initial processing feedback.  We will store your data for 1 month, then it will be queued for deletion. If you have issues downloading your data please let us know.

Your data is only accessible through the spokesperson’s login located in a few places:

1. the data on nccatweb.nysbc.org is located in /beegfs/leginon/[username]/ ,
2. the direct detector data (movies in TIFF LZW from the Gatan K3 or EER from the TFS Falcon4i) is located in /beegfs/frames/[username] ,
3. the pre-processing is in the Appion folder and may be regenerated through processing and located in /beegfs/appion/[username] , and
4. the references are located in the folder called references in the frames directory. You may see a couple because we updated the gain after we started the session. So the latest one is the one to use.

What about gain references?

For Gatan K3 images gain correction requires a y-flip for most motion correction programs (e.g. cryoSPARC). The K3 is hardware dark corrected and only the gain reference is needed. The name of the gain reference would be in a format, such as “n20dec29a_29172033_05_4092x5760_norm_0.mrc”. If there are multiple references you should use the latest. If a new gain reference was taken in the middle of the session it would be annotated in the” reference_list.txt” file.

For TFS Falcon3 images, the MRC movies in your frames folder are already dark and bright corrected. No reference files are needed for processing. If collecting on a TFS Falcon4i system, then the images would be in EER format. In this case, the references would be found in your frames folder.

For more information read our wiki [here].

We use Globus, a non-profit service for secure, reliable research data management, for data transfer.

Please note that you are responsible for your data and samples. In general, direct detector data will be queued for deletion in a month. Your Leginon images are not backed up and may be converted to JPEG versions within 1 year. We will make our best efforts to preserve the functionality of the image viewer, but do not archive data. If you are having trouble with Globus let us know. If it is the endpoint we maintain our IT may assist, but Globus is a service we subscribe to and general help may also be found on their website: https://www.globus.org/