4. RELION. We used version 3.0 [8]. Download from https://
www3.mrc-lmb.cam.ac.uk/relion.
5. A molecular volume viewer like UCSF Chimera [9] and/or
Pymol (Schro ¨dinger, LLC). We used Chimera version 1.11.2.
Download from https://www.cgl.ucsf.edu/chimera.
2.3 Test Data
1. Download the test data from the EMPIAR data base, under
accession number 10180: https://www.ebi.ac.uk/pdbe/
emdb/empiar/entry/10180.
3 Methods
3.1 Prepare
Input Files
1. Multi-body refinement starts from the so-called consensus
refinement, where all particles have been refined with respect
to a single 3D reference. Use the consensus map to define a
division of the complex into a discrete number of independently moving bodies. To ensure enough signal for accurate
alignments of the individual bodies, each body should comprise a molecular weight of at least 100–150 kDa. In the
spliceosome example, we chose to divide the complex into
four bodies: the central core, the foot domain, the helicase
domain, and the SF3b domain (Fig. 1), also see Notes 2 and 3.
2. Define a mask for each of the bodies. The masks should have a
maximum value of 1 inside the body, and a minimum value of
0 outside the body. The masks should all be on the same
Cartesian grid as the reference map of the consensus refinement, i.e., they should have the same pixel and box size as the
consensus reference. To minimize artifacts in Fourier-space,
the edges of the masks should be soft, i.e., they should gradually decrease from 1 to 0 over multiple pixels (see Note 4). The
masks for the spliceosome example are stored as Mask-andRef/*_mask.mrc.
3. Determine the order of bodies. Because earlier bodies are subtracted first, it is best to place larger and more stable bodies
earlier in the list.
4. For each body, define one neighbouring body to express how
the two bodies rotate relative to each other. This is necessary
because priors on the relative orientations between bodies are
determined along axes between the centers of mass of their
respective masks. For the spliceosome example, we chose to
have the foot, the helicase, and the SF3B domains that rotate
relative to the central core domain. The central core domain
was chosen to rotate relative to the second largest body, i.e., the
foot domain (see Note 5).
Multi-body Refinement in RELION
149
www3.mrc-lmb.cam.ac.uk/relion.
5. A molecular volume viewer like UCSF Chimera [9] and/or
Pymol (Schro ¨dinger, LLC). We used Chimera version 1.11.2.
Download from https://www.cgl.ucsf.edu/chimera.
2.3 Test Data
1. Download the test data from the EMPIAR data base, under
accession number 10180: https://www.ebi.ac.uk/pdbe/
emdb/empiar/entry/10180.
3 Methods
3.1 Prepare
Input Files
1. Multi-body refinement starts from the so-called consensus
refinement, where all particles have been refined with respect
to a single 3D reference. Use the consensus map to define a
division of the complex into a discrete number of independently moving bodies. To ensure enough signal for accurate
alignments of the individual bodies, each body should comprise a molecular weight of at least 100–150 kDa. In the
spliceosome example, we chose to divide the complex into
four bodies: the central core, the foot domain, the helicase
domain, and the SF3b domain (Fig. 1), also see Notes 2 and 3.
2. Define a mask for each of the bodies. The masks should have a
maximum value of 1 inside the body, and a minimum value of
0 outside the body. The masks should all be on the same
Cartesian grid as the reference map of the consensus refinement, i.e., they should have the same pixel and box size as the
consensus reference. To minimize artifacts in Fourier-space,
the edges of the masks should be soft, i.e., they should gradually decrease from 1 to 0 over multiple pixels (see Note 4). The
masks for the spliceosome example are stored as Mask-andRef/*_mask.mrc.
3. Determine the order of bodies. Because earlier bodies are subtracted first, it is best to place larger and more stable bodies
earlier in the list.
4. For each body, define one neighbouring body to express how
the two bodies rotate relative to each other. This is necessary
because priors on the relative orientations between bodies are
determined along axes between the centers of mass of their
respective masks. For the spliceosome example, we chose to
have the foot, the helicase, and the SF3B domains that rotate
relative to the central core domain. The central core domain
was chosen to rotate relative to the second largest body, i.e., the
foot domain (see Note 5).
Multi-body Refinement in RELION
149