You can expect your cryo-EM project to go through three main phases:
- Screening of grids
- Initial data collection
- High-resolution data collection
Every project is different, and the time you will spend on each phase of the project will depend on each unique sample and its specific requirements.
Before we can accept a sample for the Krios, the user must demonstrate the sample has an acceptable level of purity and is an appropriate target for cryo-EM. We would like to see a gel filtration profile and Comassie gel image for the sample, and a characteristic negative stain image. Before starting you will need to submit a project form. If you are not sure your project is appropriate for cryo-EM, please contact Claire for more information about project design by emailing email@example.com
Screening of Grids
In this phase of the project you will plunge freeze your sample. Often we will try different freezing conditions and/or different protein concentrations to determine the optimum conditions for your sample. In a microscope screening session up to 11 grids will be loaded onto the microscope and we will look at them to assess ice quality, appropriate protein concentration, and protein behaviour. This can often be an iterative process that involves the user re-optimising the sample for purity, and/or trying different freezing conditions. The user is expected to be present during the microscope screening sessions.
Initial Data Collection
Once we have found freezing conditions that appear promising, we will collect an initial data set. This will usually be an overnight collection session of ~10-14 hours. This data set will be collected at a magnification of around 47,000x, giving the user a sufficient number of particles for an initial analysis. This can reveal heterogeneity in the sample, and/or provide an initial model for the sample. If the analysis yields a high-quality initial model, this indicates the sample is a good candidate for high-resolution structural characterisation.
High-resolution Data Collection
Once we are sure the sample is of good enough quality, we will set up a longer data collection at a higher magnification. The aim of this is to get a large number of particles that contain high-resolution data which can then be used to reconstruct a high resolution structure. These data collection sessions will typically span several days. While the data collection is automated, the user is responsible for determining which areas to collect on, and be available throughout the collection session for consultation.