5. Repeat steps 2–4 for the remaining three bodies.
6. Inspect the Fourier Shell Correlation (FSC) plot (Fig. 3) in the
“logfile.pdf” from the “Display!” pull-down menu for
each of the four bodies. Confirm that the red FSC curve is close
to zero at the resolution estimate of each body. If this is not the
case, their resolution estimate may be unreliable due to your
masks being too tight or too sharp (see Note 17). In that case,
repeat multi-body refinement with wider and/or softer-edged
masks.
7. The post-processed maps of the four bodies obtained in steps
2–5 can be opened in molecular volume viewers and used for
separate atomic modeling of the individual bodies, for example,
in COOT [10] (see Notes 18 and 19).
3.4 Analyze the Body
Motions
1. Select “MultiBody/job001” in the “Finished jobs” window on the lower part of the GUI. Use the “Display:” dropdown menu to visualize the “analyse_logfile.pdf” file.
The first plot (Fig. 4a) shows how much variance in the orientations is explained by each of the principal components. The
subsequent plots show histograms of the projections of the
relative orientations of the bodies of each experimental projection image onto the corresponding component. Unimodal
histograms are an indication of continuous motions in the
complex, whereas bi- or multi-modal histograms indicate the
presence of two or more discrete states. In the spliceosome
example, all histograms are unimodal, but in other cases it may
be useful to select subsets of particles based on their projections
along the principal components (see Note 20).
2. Make a movie that represents this first principal motion. Ten
maps called “analyse_component001_bin0??.mrc” are
generated by rotating and translating each body along each
principal motion. In Chimera, from the “Tools” menu, select
“Volume Data” and then “Volume Series”. Click on the
“Open...” button and browse to the directory “Multibody/job001”. To make a movie for the first principal component, hold down the SHIFT key and use the left-mouse
button to select all ten maps called “analyse_component001_bin0??.mrc”. Then, change “Play direction”
to “oscillate” and press “Play”. You can change the sampling rate and threshold of the maps through the “Volume
Viewer” tool, or by using the “Command Line” from the
“Favourites” menu, and typing: “vol all step 2” and
“vol all level 0.015” (see Note 21).
3. Write a movie to file by selecting from the “Tools” menu
“Utilities” and then “Movie recorder”.
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153
6. Inspect the Fourier Shell Correlation (FSC) plot (Fig. 3) in the
“logfile.pdf” from the “Display!” pull-down menu for
each of the four bodies. Confirm that the red FSC curve is close
to zero at the resolution estimate of each body. If this is not the
case, their resolution estimate may be unreliable due to your
masks being too tight or too sharp (see Note 17). In that case,
repeat multi-body refinement with wider and/or softer-edged
masks.
7. The post-processed maps of the four bodies obtained in steps
2–5 can be opened in molecular volume viewers and used for
separate atomic modeling of the individual bodies, for example,
in COOT [10] (see Notes 18 and 19).
3.4 Analyze the Body
Motions
1. Select “MultiBody/job001” in the “Finished jobs” window on the lower part of the GUI. Use the “Display:” dropdown menu to visualize the “analyse_logfile.pdf” file.
The first plot (Fig. 4a) shows how much variance in the orientations is explained by each of the principal components. The
subsequent plots show histograms of the projections of the
relative orientations of the bodies of each experimental projection image onto the corresponding component. Unimodal
histograms are an indication of continuous motions in the
complex, whereas bi- or multi-modal histograms indicate the
presence of two or more discrete states. In the spliceosome
example, all histograms are unimodal, but in other cases it may
be useful to select subsets of particles based on their projections
along the principal components (see Note 20).
2. Make a movie that represents this first principal motion. Ten
maps called “analyse_component001_bin0??.mrc” are
generated by rotating and translating each body along each
principal motion. In Chimera, from the “Tools” menu, select
“Volume Data” and then “Volume Series”. Click on the
“Open...” button and browse to the directory “Multibody/job001”. To make a movie for the first principal component, hold down the SHIFT key and use the left-mouse
button to select all ten maps called “analyse_component001_bin0??.mrc”. Then, change “Play direction”
to “oscillate” and press “Play”. You can change the sampling rate and threshold of the maps through the “Volume
Viewer” tool, or by using the “Command Line” from the
“Favourites” menu, and typing: “vol all step 2” and
“vol all level 0.015” (see Note 21).
3. Write a movie to file by selecting from the “Tools” menu
“Utilities” and then “Movie recorder”.
Multi-body Refinement in RELION
153