Q: What symmetry should I specify during refinement if I don't know what it is ?
A: There is no single good answer to this question. It can be a very complicated issue. Take the case of TriC/CCT, for example:
http://www.pnas.org/content/107/11/4967.abstract
In this case, the object in question really has no symmetry, or at most D1 symmetry, since it is constructed of 8 unique proteins. However, these proteins have extremely high homology, and in fact, at resolutions worse than 4-6 A, the structure effectively has D8 symmetry, because the differences only emerge at higher resolutions. Even for particles which don't have different proteins, the concept of true symmetry at all resolutions is a myth. At SOME resolution the symmetry will be broken, but this symmetry breaking resolution may have any value.
Subtle symmetry arguments aside, for most symmetric particles, the symmetry can be fairly easily observed in reference-free class averages, so this is a good place to start when assessing the symmetry of an object. Of course, it is possible that even if your object is symmetric, the symmetric view of the object may be absent in your particle set due to problems with preferred orientation. This can make accurate symmetry assessment much more challenging if true.
So, look at your reference-free class averages and consider the following possibilities:
- icos : icosahedral symmetry. You should have 5-folds, 3-folds and 2-folds, though often all won't be readily seen. Generally the objects will be ball-like, such as virus capsids.
- oct : octahedral symmetry. You should see 4-folds, 3-folds and 2-folds. This is the symmetry of a cube or an octahedron.
- tet : tetrahedral or cubic symmetry. 3-folds and 2-folds (no 4-fold). This is the symmetry of a tetrahedron.
- Cn : A single rotational symmetry axis with n-fold symmetry.
- Dn : Same as Cn but with a 2-fold symmetry 90 degrees from the Cn axis. Generally this would mean 2 ring-like structures attached to each other, for example most chaperonins, apoptosome, etc. Observed as a class-average with an n-fold symmetry, and another class-average with a clear 2-fold symmetry (usually also pseudo-mirror symmetry)
- C1 : No symmetry. In fact, you can simply omit the sym= option in EMAN2 programs, but if you need to specify something, just say C1.
Once a putative symmetry has been identified, you can try running e2initialmodel.py using this symmetry on your class-averages, and assess the results. This program is relatively fast, so you can try different options if more than one possibility exists. Once you have a reasonable guess at the symmetry, try using this symmetry for a full refinement using your raw data.
Once you have a self-consistent refinement, you can decide whether your symmetry assessment was correct. If you are concerned that you may still be imposing the incorrect symmetry, you can try relaxing the symmetry, by starting with a symmetric starting model, but using C1 symmetry for the refinement. If you had the incorrect symmetry it is likely that within a few rounds of refinement, the reconstruction will degrade substantially. If you had the correct symmetry, the symmetry will still be broken during C1 refinement due to model bias, but the symmetry breaking should be more subtle, and will generally involve things like linear density gradients across the reconstruction.