Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION
- MRC Laboratory of Molecular Biology, United Kingdom
- Stockholm University, Sweden
- Swedish e-Science Research Center, KTH Royal Institute of Technology, Sweden
Figures
Figure 1 with 2 supplements
A schematic overview of multi-body refinement.
After a consensus 3D auto-refinement in RELION, the three-dimensional consensus map is split into separate bodies using user-defined masks. In this example, and the letters ’M’ (orange), ’R’ …
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Figure 1—source data 1
The body definition STAR file.
Example of a STAR file that defines the bodies in Figure 1. The column labelled rlnBodyMaskName defines the file names for the corresponding masks; the relative orientations of eachbody are defined by the columns (rlnBodyRotateRelativeTo); and the priors on the rotations and translations of the bodies are given by rlnBodySigmaAngles and rlnBodySigmaOffset, respectively.
- https://doi.org/10.7554/eLife.36861.005
Figure 1—figure supplement 1
Overlapping body masks.
(A) Overlapping masks for the example shown in Figure 1. For the partial signal subtraction for focused refinement of the ’M’ body, the density of the ’R’ body within the ’R’ mask but not within the …
Figure 1—figure supplement 2
Relative body orientations.
The density for each body is placed with the centre-of-mass of its mask (indicated with orange and green dots for the ’M’ body and ’R’ body, respectively) at the centre of the three-dimensional box, …
Figure 2 with 1 supplement
The ribosome test case.
(A) The ribosome consensus map with the three transparent body masks (LSU, SSU and head) superimposed. (B) Slices through the density of the three bodies after the consensus refinement (top) and …
Figure 2—figure supplement 1
Fourier shell correlation curves calculated from independently refined halves of the data for the three bodies after consensus refinement (dashed lines) and after the second multi-body refinement (solid lines).
https://doi.org/10.7554/eLife.36861.007
Figure 3 with 3 supplements
The spliceosome test case.
(A) The four body masks used for the first splicesome multi-body refinement are shown in semi-transparent colours on top of the consensus map on the left; the resulting density after the first …
Figure 3—figure supplement 1
Fourier shell correlation curves calculated from independently refined halves of the data for the four bodies after consensus refinement (dashed lines), for the largest class of a discrete 3D classification (dotted lines) and after the second multi-body refinement (solid lines).
https://doi.org/10.7554/eLife.36861.011
Figure 3—figure supplement 2
Local resolution estimates (in Å) for the SF3b region after multi-body refinement (left) and after subsequent partial signal subtraction in relion_flex_analyse followed by focused classification and refinement of the best class (right).
https://doi.org/10.7554/eLife.36861.012
Figure 3—figure supplement 3
The largest two classes of a 3D classification with eight classes (right) represent a similar motion as identified by the first principal component from the multi-body approach (left).
For the latter, the third and eight out of ten movie frames are shown.
Videos
Video 1
Repositioning of the reconstructed body densities along the first eigenvector for the ribosome case reveals a rolling-like motion of the SSU with respect to the LSU and a concomitant swiveling of the head.
https://doi.org/10.7554/eLife.36861.008
Video 2
Repositioning of the reconstructed body densities along the second eigenvector for the ribosome case reveals a ratchet-like motion of the SSU with respect to the LSU together with a displacement of the head.
https://doi.org/10.7554/eLife.36861.009
Video 3
Repositioning of the reconstructed body densities along the first eigenvector for the spliceosome case reveals a rocking motion of the SF3b domain over the core.
https://doi.org/10.7554/eLife.36861.014
Video 4
Repositioning of the reconstructed body densities along the second eigenvector for the spliceosome case reveals a concerted rocking motion of the helicase and SF3b over the core.
https://doi.org/10.7554/eLife.36861.015Tables
Key resources table
| Reagent type | Designation | Reference | Identifier |
|---|---|---|---|
| software | relion | (Scheres, 2012b) | RRID:SCR_016274 |
