following input: (a) candidate model pdb file, (b) EM density map
file, (c) map resolution. The type of fitting (local/global) can be
specified by modifying the grid_search method (see Note 7). More
details can be found here.
5.2.1 Finding Rigid
Bodies with RIBFIND
To use RIBFIND, a web server has been set up (see Note 8)
(although the standalone program can also be downloaded). RIBFIND accepts the following inputs: (a) the protein coordinates in
PDB file format (a single chain or multiple chains with the residues
numbered continuously); (b) a description of the protein SSEs in
DSSP [20] format (optional, if not provided, the server automatically runs DSSP); (c) the value of the contact distance parameter;
(d) the email address of the user; and (e) a density map in MRC
format (optional). The RIBFIND client interface reads the input,
validates it, and submits it to the server for execution. After completing the job, the server sends an automatic email to the user with
a link to the results page. This page contains a NGL Viewer applet
showing a cartoon of the user PDB file with each rigid-body
uniquely colored. All SSEs and loops that are not part of a cluster
are colored white. The rigid-body set that is displayed by default is
the one that contains the maximal number of clusters identified by
RIBFIND (we previously showed that using this set in cryo-EM
flexible fitting produced the best results in most cases [42]). Using
the slider control on the results page, the user can view different
sets of rigid bodies generated for each cluster cutoff and save the
corresponding rigid-body file in a text format which can be used for
refinement, e.g., by Flex-EM [5].
5.3 TEMPy
TEMPy is a python library that allows the user to design bespoke
pipelines for the manipulation and analysis of candidate models and
EM maps [10, 11, 16, 17]. The software package and documentation can be found here: http://tempy.ismb.lon.ac.uk/. The protocols for some commonly used functions are outlined here.
5.3.1 Structure Blurring
Blurred maps derived from candidate models are needed for the
analysis of the goodness-of-fit of proposed candidate models into
density maps. To blur a map, TEMPy requires (a) the PDB or
mmCIF file representing the coordinates and atom types of the
candidate model; (b) the resolution (A ˚ ) to blur the protein to; and
(c) the sigma value (multiplied by the resolution) that controls the
width of the Gaussian (with default value of 0.356). An additional
option “DensMap” controls whether the blurred map dimensions
are based on the candidate structure or the EM map.
5.3.2 Scoring Fits
TEMPy offers a wide range of global and local scoring functions to
analyze the goodness-of-fit, both globally and locally. Depending
on the map resolution, extent of overlap or shape features, one
scoring function may be more useful than others. Both CCC and
CryoEM Density Fitting and Validation
213
file, (c) map resolution. The type of fitting (local/global) can be
specified by modifying the grid_search method (see Note 7). More
details can be found here.
5.2.1 Finding Rigid
Bodies with RIBFIND
To use RIBFIND, a web server has been set up (see Note 8)
(although the standalone program can also be downloaded). RIBFIND accepts the following inputs: (a) the protein coordinates in
PDB file format (a single chain or multiple chains with the residues
numbered continuously); (b) a description of the protein SSEs in
DSSP [20] format (optional, if not provided, the server automatically runs DSSP); (c) the value of the contact distance parameter;
(d) the email address of the user; and (e) a density map in MRC
format (optional). The RIBFIND client interface reads the input,
validates it, and submits it to the server for execution. After completing the job, the server sends an automatic email to the user with
a link to the results page. This page contains a NGL Viewer applet
showing a cartoon of the user PDB file with each rigid-body
uniquely colored. All SSEs and loops that are not part of a cluster
are colored white. The rigid-body set that is displayed by default is
the one that contains the maximal number of clusters identified by
RIBFIND (we previously showed that using this set in cryo-EM
flexible fitting produced the best results in most cases [42]). Using
the slider control on the results page, the user can view different
sets of rigid bodies generated for each cluster cutoff and save the
corresponding rigid-body file in a text format which can be used for
refinement, e.g., by Flex-EM [5].
5.3 TEMPy
TEMPy is a python library that allows the user to design bespoke
pipelines for the manipulation and analysis of candidate models and
EM maps [10, 11, 16, 17]. The software package and documentation can be found here: http://tempy.ismb.lon.ac.uk/. The protocols for some commonly used functions are outlined here.
5.3.1 Structure Blurring
Blurred maps derived from candidate models are needed for the
analysis of the goodness-of-fit of proposed candidate models into
density maps. To blur a map, TEMPy requires (a) the PDB or
mmCIF file representing the coordinates and atom types of the
candidate model; (b) the resolution (A ˚ ) to blur the protein to; and
(c) the sigma value (multiplied by the resolution) that controls the
width of the Gaussian (with default value of 0.356). An additional
option “DensMap” controls whether the blurred map dimensions
are based on the candidate structure or the EM map.
5.3.2 Scoring Fits
TEMPy offers a wide range of global and local scoring functions to
analyze the goodness-of-fit, both globally and locally. Depending
on the map resolution, extent of overlap or shape features, one
scoring function may be more useful than others. Both CCC and
CryoEM Density Fitting and Validation
213