3D Variabilityjob in cryoSPARC v2.9+.
3D Variabilityjob when you suspect or are interested in investigating conformational variability in your dataset. Through this tutorial and the example results below, we can conclude that many molecules analyzed through single particle cryoEM contain at least some observable 3D variability.
2D Classification. The goal is to remove obvious "junk" particles that may later affect the quality of reconstruction and refinement, but not to try and separate conformations.
Number of Ab-initio classes≥ 2) in order to find and exclude grossly different structures that may be present in the data set (e.g., if you have two completely different molecules present). You may also observe some heterogeneity that is conformational variability of a single molecule (e.g,. a subunit moving/flexing or portions dissociating, etc.) - this is fine.
Refinement box size (Voxels)to the full resolution/box size of the particles. If this is very large, it is better to use the
Downsample Particlesjob first to downsample the particles, do the consensus refinement again, and then apply 3D variability analysis. During the refinement, optionally enter a value to enforce
Symmetry(e.g., C4) if known.
3D Variabilityjob and connect both the particles and the mask from the previous refinement.
3D Variabilityjob to perform "focused variability".
Number of modes to solve: This is the number of components/eigenvectors that will be solved for. The default value is 3, and the maximum is limited by GPU memory and the box size. Generally more modes will take (linearly) more time to solve. One subtle but important point is that the algorithm will generally solve these in order of importance, so asking for more modes will not reduce the quality of the first, most important modes of variability. NB. in v2.9 there is a known bug where sometimes, due to numerical instability, using more modes can result in "streaking" and other artefacts in the results. We are working on fixing this.
Filter resolution (Å): This is the resolution at which eigenvectors are filtered during optimization. We recommend setting this to a value lower than the resolution of the consensus refinement performed previously (e.g., you may set it to 5A for a 3A refinement, but it can be experimented with). Using too high of a resolution will allow noise to dominate the determination of the eigenvectors.
3D Variability(as well as the preceding refinement) must be run at the full resolution/box size of the particles. You can downsample particles before processing, or else downsample the results before downloading outputs, in the next step. Also, you cannot currently enforce symmetry during this job.
3D Variability Displayjob, connecting the particles and volumes from the
Output mode: There are several output modes that can be used for different purposes. NB. in v2.9.0 only the simple mode is exposed as the others are experimental. Simple mode: This will output a simple linear "movie" of volumes along each eigenvector. You can control the number of frames, and the starting/ending position as a percentile of the reaction coordinate distribution. The output will be a set of "volume series" which are essentially just a set of numbered
.mrcfiles that can be opened with Chimera (see below).
Number of frames: How many frames (per dimension) or clusters to output.
Downsample to box size: Downsample the volumes before writing, to save disk space.
Crop to size (after downsample): Crop the volumes after downsampling, to save disk space.
3D Variability Displaywill output a series of frames for each component of the variability detected in the dataset. The outputs can be used to either download and view as movies in Chimera, or can be used to provide initial models and particle subsets for further classification and refinement. See the Visualization section below for detailed instructions about how to display the results in Chimera. Also see the second part of this tutorial series for more advanced usage and interpretation.
frame_XXX.mrcfiles from a single component. This will open them all together as a series.
set bgColor whiteand a colour of