You have been assigned a Grid Preparation and Screening (GPS) session at NCCAT. What are the next steps?
Sample or grid requirements:
1. If sending samples:
The amount of sample required for 3-4 days of screening and optimization largely depends on the sample itself, the types of challenges it faces, the optimization steps required. As a ballpark starting point, we recommend:
Sample concentration: > 2 mg/ml
Sample volume: > 1 ml
Buffer volume: 100 ml
This is a general recommendation aimed to allow for trying as many screening conditions as possible in 3-4 days, and not a rule. We will work with you and your sample to determine a suitable amount of samples to prepare for the GPS session.
NCCAT provides common cryo-EM consumables. Please ensure you ship or bring non-cryo-EM or bespoke cryo-EM consumables you might need.
2. If sending grids:
You may send up to 24 grids in 6 boxes. Your grids will be automatically screened on our NCCAT Glacios with Smart Leginon, with a maximum of one cassette loading in the morning and one in the evening.
1. If you sent samples:
A customized three- or four-day plan for screening and optimizing your samples will be developed together with you during your pre-session meeting.
A staff member will work closely with you when you’re on site to screen and optimize your sample for cryo-EM. In general, we will be aiming for the following workflow:
1. Prepare sample with generic grid-preparation conditions.
2. Image sample on a TEM and analyze images to understand potential issues.
3. Optimize sample preparation conditions.
4. Repeat steps 2-3.
From experience, grid-making conditions for 1-2 samples can be optimized in this 3 to 4-day period, during which 15~30 grids are likely to be prepared under various conditions, depending on the needs of the sample.
You will have access to all the sample optimization tools available on-site, including carbon and gold grids, graphene grids, Vitrobot, the Chameleon, and Refeyn. We also have a buffer optimization screening kit, and a fully equipped wet lab. NCCAT provides common cryo-EM consumables; please ensure you ship or bring non-cryo-EM or bespoke cryo-EM consumables you might need.
In a three-day visit, we will aim to complete three cycles of the screening workflow with the goal of preparing Krios-ready grids. At the end of your visit, we will review the findings together and put together a plan for next steps. If more biochemical optimization of your sample or data processing at your home institution is required to improve or better understand the sample, one supplementary visit to NCCAT can be arranged once the optimization and processing steps are complete. There will be another pre-session meeting ahead of the supplementary visit to plan resources and strategize.
2. If you sent grids:
Your grids will be automatically screened on our NCCAT Glacios microscope with Smart Leginon. Your data can be viewed by using the Leginon and Appion webtools as detailed below. You can choose to review your data with us after the session.