molecules. If the model is outside the map, it must be roughly
placed into the map first.
6.5 Rigid-Body
Fitting
Note 7: Mod-EM scripts are available from https://salilab.org/
modeller/tutorial/cryoem/fit.html. Documentation on modeler
density and grid_search classes are available here: https://salilab.
org/modeller/9.21/manual/
6.6 RIBFIND
Note 8: RIBFIND web server available at http://ribfind.ismb.lon.
ac.uk/
6.7 Flex-EM
Note 9: Flex-EM scripts are available at http://topf-group.ismb.
lon.ac.uk/flex-em/
Note 10: If the rigid bodies are defined at the domain level, and
a relatively large conformational change is expected (e.g., an ion
channel moving from open to closed state) the recommended cap
shift is 0.39 A ˚ . If rigid bodies are defined at the SSE level, the
recommended cap shift is 0.15 A ˚ and 0.1 A ˚ for all atom level.
Note 11: Initial fits can be achieved in Chimera using the “fit in
map” tool and saving the PDB with new coordinates relative to the
input density map.
Note 12: The X, Y, Z origins can be found in Chimera using the
“volume viewer” command. If the origin is given in pixels (e.g.,
from Chimera), the values are calculated as À1 * voxel size.
Note 13: The output of RIBFIND can be used directly as input
for Flex-EM. Additionally, custom rigid-body files can be created.
The format of these files are: lines starting with # are comments
(e.g., #Helix), subsequent lines define a single rigid body. Residues
contributing to a rigid bodies are defined by the start and stop
residue (e.g., 24 33) multiple segments can be assigned to a single
rigid body (e.g., “2 6 28 30” means that residues 2–6 and 28–30
will be included in the same rigid body). For multi-chain models
residues can be specified within chains (e.g., “2:A 6:A 28:B 30:B”
means that residues 2–6 from chain A and 28–30 from chain B will
be included in the same rigid body). For including ligands and
other hetero-atoms (HETATM), the molecule number and chain
ID can be added to the rigid-body line in the same format (but it
should not form a separate rigid body, e.g., “100:A 100:A” means
that the HETATM 100 from chain A will be considered as a
rigid body).
6.8 Difference Maps
Note 14: If the input is given as two maps, both maps must first be
aligned. This can be done automatically in Chimera using the “fit in
map” tool. However, if it is expected that the two maps represent
vastly different conformations (e.g., a large conformational change
upon ligand binding) the optimum fit may not be correct” in this
CryoEM Density Fitting and Validation
219
placed into the map first.
6.5 Rigid-Body
Fitting
Note 7: Mod-EM scripts are available from https://salilab.org/
modeller/tutorial/cryoem/fit.html. Documentation on modeler
density and grid_search classes are available here: https://salilab.
org/modeller/9.21/manual/
6.6 RIBFIND
Note 8: RIBFIND web server available at http://ribfind.ismb.lon.
ac.uk/
6.7 Flex-EM
Note 9: Flex-EM scripts are available at http://topf-group.ismb.
lon.ac.uk/flex-em/
Note 10: If the rigid bodies are defined at the domain level, and
a relatively large conformational change is expected (e.g., an ion
channel moving from open to closed state) the recommended cap
shift is 0.39 A ˚ . If rigid bodies are defined at the SSE level, the
recommended cap shift is 0.15 A ˚ and 0.1 A ˚ for all atom level.
Note 11: Initial fits can be achieved in Chimera using the “fit in
map” tool and saving the PDB with new coordinates relative to the
input density map.
Note 12: The X, Y, Z origins can be found in Chimera using the
“volume viewer” command. If the origin is given in pixels (e.g.,
from Chimera), the values are calculated as À1 * voxel size.
Note 13: The output of RIBFIND can be used directly as input
for Flex-EM. Additionally, custom rigid-body files can be created.
The format of these files are: lines starting with # are comments
(e.g., #Helix), subsequent lines define a single rigid body. Residues
contributing to a rigid bodies are defined by the start and stop
residue (e.g., 24 33) multiple segments can be assigned to a single
rigid body (e.g., “2 6 28 30” means that residues 2–6 and 28–30
will be included in the same rigid body). For multi-chain models
residues can be specified within chains (e.g., “2:A 6:A 28:B 30:B”
means that residues 2–6 from chain A and 28–30 from chain B will
be included in the same rigid body). For including ligands and
other hetero-atoms (HETATM), the molecule number and chain
ID can be added to the rigid-body line in the same format (but it
should not form a separate rigid body, e.g., “100:A 100:A” means
that the HETATM 100 from chain A will be considered as a
rigid body).
6.8 Difference Maps
Note 14: If the input is given as two maps, both maps must first be
aligned. This can be done automatically in Chimera using the “fit in
map” tool. However, if it is expected that the two maps represent
vastly different conformations (e.g., a large conformational change
upon ligand binding) the optimum fit may not be correct” in this
CryoEM Density Fitting and Validation
219