Servalcat

Structure refinement and validation for crystallography and single particle analysis

Currently we focus on development for single particle analysis and there are only a few functions at the moment.

Installation

pip install servalcat

will install the stable version.

If you want to try the latest features,

pip install git+https://github.com/keitaroyam/servalcat.git

Add -U option for updating.

The required GEMMI version is now v0.5.8. Please update GEMMI as well if it is old (it should be done by pip).

Usage

servalcat <command> <args>

The most useful commands are shown below. To see all arguments for each command please run

servalcat <command> -h

Please also read the documentation: https://servalcat.readthedocs.io/en/latest/

Refinement using REFMAC5

Servalcat makes refinement by REFMAC5 easy for single particle analysis. The weighted and sharpened Fo-Fc map is calculated after the refinement. For details please see the reference.

Make a new directory and run:

servalcat refine_spa \
 --model input.pdb --resolution 2.5 \
 --halfmaps ../half_map_1.mrc ../half_map_2.mrc \
 --ncycle 10 [--pg C2] \
 [--mask_for_fofc mask.mrc]

Specify unsharpened and unweighted half maps (e.g. those after Refine3D of RELION) after --halfmaps.

If map has been symmetrised with a point group, asymmetric unit model should be given together with --pg to specify a point group symbol. It assumes the center of the box is the origin of the symmetry and the axis convention follows RELION.

Other useful options:

• --ligand lig.cif : specify restraint dictionary (.cif) file(s)
• --mask_for_fofc mask.mrc : speify mask file for Fo-Fc map calculation
• --jellybody : turn on jelly body refinment
• --weight_auto_scale value : specify weight auto scale. by default Servalcat determines it from resolution and mask/box ratio
• --keyword_file file : specify any refmac keyword file(s) (e.g. prosmart restraint file)
• --pixel_size value : override pixel size of map
• --exe refmac5 : specify REFMAC5 binary

Output files:

• refined.pdb: refined model
• refined_expanded.pdb: symmetry-expanded version
• diffmap.mtz: can be auto-opened with coot. sharpened and weighted Fo map and Fo-Fc map
• diffmap_normalized_fofc.mrc: Fo-Fc map normalized within a mask. Look at raw values
• shifted_refined.log: refmac log file

Fo-Fc map calculation

It is important to refine individual atomic B values with electron scattering factors to calculate meaningful Fo-Fc map. Fo-Fc map is calculated in refine_spa command (explained above) so usually you do not need to run fofc command manually, but you may want to calculate e.g. omit maps.

servalcat fofc \
 --model input.pdb --resolution 2.5 \
 --halfmaps ../half_map_1.mrc ../half_map_2.mrc \
 [--mask mask.mrc] [-o output_prefix] [-B B value]

-B is to calculate weighted maps based on local B estimate. It may be useful for model building in noisy region.

Map trimming

Maps from single particle analysis often have very large size due to unnccesary region outside the molecule. You can save disk space by trimming the unnccesary region.

servalcat trim \
 --maps postprocess.mrc halfmap1.mrc halfmap2.mrc \
 [--mask mask.mrc] [--model model.pdb] [--padding 10]

Maps specified with --maps are trimed. The boundary is decided by --mask or --model if mask is not available. Model(s) are shifted into a new box. By default new boundary is centered on the original map and cubic, but they can be turned off with --noncentered and --noncubic. If you do not want to shift maps and models, specify --no_shift to keep origin.

Reference

Yamashita, K., Palmer, C. M., Burnley, T., Murshudov, G. N. (2021) "Cryo-EM single particle structure refinement and map calculation using Servalcat" Acta Cryst. D77, 1282-1291