| |
| eman2:tomosymmetry [2025/06/19 01:20] – created steveludtke | eman2:tomosymmetry [2025/06/19 01:21] (current) – steveludtke |
|---|
| |
| Symmetric biomolecules really aren't (symmetric). At some limiting resolution, the symmetry will always be broken. Consider 3 examples: | Symmetric biomolecules really aren't (symmetric). At some limiting resolution, the symmetry will always be broken. Consider 3 examples: |
| * small rigid enzyme - symmetry breaking might be at the level of independent individual sidechain motion, may only impact at 3-5 � resolution | * small rigid enzyme - symmetry breaking might be at the level of independent individual sidechain motion, may only impact at 3-5 Å resolution |
| * C4 ion channel - Each of the 4 subunits will somewhat independently bind modulators or be otherwise conformationally variable, while maintaining overall symmetry, may impact at the level of 6-12 � resolution | * C4 ion channel - Each of the 4 subunits will somewhat independently bind modulators or be otherwise conformationally variable, while maintaining overall symmetry, may impact at the level of 6-12 Å resolution |
| * C8 nuclear pore complex - While ostensibly 8-fold (some parts D8) symmetric, in reality, the variations in separation of the inner and outer nuclear membranes, interactions with substrates, etc, require significant accommodations by the complex, resulting in large 20-40 � resolution symmetry breaking | * C8 nuclear pore complex - While ostensibly 8-fold (some parts D8) symmetric, in reality, the variations in separation of the inner and outer nuclear membranes, interactions with substrates, etc, require significant accommodations by the complex, resulting in large 20-40 Å resolution symmetry breaking |
| |
| In canonical single particle reconstruction, you would simply impose the specified symmetry and hope for the best. Clearly, however, the structure resulting from a symmetrized average of non-identical or mispositioned components will not result in the best possible subunit resolution. We instead need a strategy to look at a single subunit at a time, and refine that, characterizing/classifying not entire symmetric particles, but on an individual subunit basis. | In canonical single particle reconstruction, you would simply impose the specified symmetry and hope for the best. Clearly, however, the structure resulting from a symmetrized average of non-identical or mispositioned components will not result in the best possible subunit resolution. We instead need a strategy to look at a single subunit at a time, and refine that, characterizing/classifying not entire symmetric particles, but on an individual subunit basis. |
| 11. Make a mask for a single subunit from the previous refinement results. | 11. Make a mask for a single subunit from the previous refinement results. |
| * The //mask.cylinder// processor can be quite useful in generating suitable alignment masks for C and D symmetric objects. It is a good idea to give the mask a soft edge either by low pass filtering it (Gaussian blur) or using the //tri// parameters in //mask.cylinder//. //e2filtertool.py// is a very useful tool when fine tuning the parameters of such masks. | * The //mask.cylinder// processor can be quite useful in generating suitable alignment masks for C and D symmetric objects. It is a good idea to give the mask a soft edge either by low pass filtering it (Gaussian blur) or using the //tri// parameters in //mask.cylinder//. //e2filtertool.py// is a very useful tool when fine tuning the parameters of such masks. |
| * Alternatively, you can do something like use the //segger// module in Chimera to extract a single subunit from the volume, then use a sequence of //filter.lowpass.gauss// and //threshold.binary// operations to turn the extracted subunit into an appropriate subunit mask which more closely follows the shape of the actual assembly. If you do this, make sure that the mask is at least 10-20 � larger than the surface of the subunit, and has a soft edge. | * Alternatively, you can do something like use the //segger// module in Chimera to extract a single subunit from the volume, then use a sequence of //filter.lowpass.gauss// and //threshold.binary// operations to turn the extracted subunit into an appropriate subunit mask which more closely follows the shape of the actual assembly. If you do this, make sure that the mask is at least 10-20 Å larger than the surface of the subunit, and has a soft edge. |
| * When making alignment masks it is **always critical** that the edge of the mask have a "soft" Gaussian edge, with a with of at least 1.5 - 2x the target resolution. For example, if you are targeting 4 � resolution, a binary mask should be lowpass filtered to at least 8 � before using it for alignment. | * When making alignment masks it is **always critical** that the edge of the mask have a "soft" Gaussian edge, with a with of at least 1.5 - 2x the target resolution. For example, if you are targeting 4 Å resolution, a binary mask should be lowpass filtered to at least 8 Å before using it for alignment. |
| |
| ---- | ---- |
