LocalFSC

LocalFSC is a plugin for Chimera to estimate the resolution in selected subregions of a EM map.

The resolution is estimated by means of the Fourier Shell Correlation (FSC), calculated from two independent maps. Generally these are the maps each generated from half the particles in a data set. Selection is performed by placing markers in the regions of interest, and the size of the regions to analyse can be set either manually or automatically. Comments can be added to each measure, and all the estimates can be saved in a text file for further analysis.

• If you don't already have a directory on your computer that is specific for external plugins for chimera, create one.
• Download the file LocalFSC_chimera.tar.gz in this directory and uncompress it.
• In Chimera, go to Favorites→Preferences, from Category select Tools and add the directory containing the LocalFSC folder to the Locations.

The LocalFSC module will be under the Tools→Volume Data menu.

Once opened, the LocalFSC module, a help button provides all the basic information on how to use it and on all the different options available in the interface.

In order to get familiar with the module, here is provided a Test Dataset to download, which contains a simulated reconstruction of a ribosome (see article below for details). Specifically, the archive file contains three maps (two half maps and one map from all the particles) and a Chimera marker file with two markers pointing to two domains with different occupancy level.

• Uncompress the data.
• Open the tree maps and the marker file in Chimera.
• Verify that all three maps are overlapping and their pixel size is properly set in the Features→Coordinates menu of the Volume Viewer dialog window.
• Hide the two half maps.
• Open the module (Tools→Volume Data→LocalFSC in the main window or Tools→LocalFSC in Volume Viewer).
• In Settings, point the first and second map to the two half maps.
• Still in Settings, set the maximum resolution available (i.e. unfiltered out) in the half maps (in this case 10).
• In the main window, select one of the markers with Ctrl-left click and the press the Estimate button in Resolution. The estimate will be listed in the GUI, and the FSC plot will be shown in a separate window.
• You can select multiple markers where to estimate the resolution. In this case the program will not show their corresponding FSC plots.

The plugin was developed by Giovanni Cardone. Acknowledgements go to Tom Goddard, UCSF, for revising the code and providing useful insights.

If you find this plugin useful for your research project, please cite:

Cardone G., J. B. Heymann, and A. C. Steven (2013) One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184:226-236. (http://dx.doi.org/10.1016/j.jsb.2013.08.002)