The road to a GOOD Cryo-EM structure

The road to a GOOD Cryo-EM structure, i.e. a high‐quality structure that will lead to a
good story.

1. Initial evaluation: what questions you expect to answer based on the structure;
what level of detail is needed; any atomic models available for part of the
protein complex; any homology models available; any binding partners that may
help improve the homogeneity.

2. Understand the intrinsic properties of the protein: size and symmetry may be
something to be considered, but there is no simple criterion. Therefore, it’s
important to have a deep understanding of all the biochemical properties of the
target protein.

3. Optimize the protein biochemically: identify the most promising isoform,
expression system, purification method, essential co‐factors, etc., evaluated by
various biochemical benchmarks and negative staining EM.

4. Make the protein amenable to Cryo-EM: Luck and commitment. Do not expect it
to be much easier than optimizing crystals.

5. Collect data and generate 3D reconstructions: while there are various data
processing tools/tricks to try, when none of them give the desired results, then
it’s necessary to go back to step 3 or 4.

6. Validate the results: whether the reconstruction and model are correct. Lower
resolution usually requires more extensive validation, including biochemical
approaches (e.g. cross‐linking, mutation).

7. Corroborate the findings based on the structure with functional studies: another
level of validation. Whether the particular construct is functional, how the
treatment during purification and Cryo-EM sample preparation affects the
physiological relevance.