servalcat

name	servalcat
description	Servalcat for cryo-EM SPA reciprocal-space refinement and Fo-Fc map calculation. Use for refining models against half-maps, calculating weighted difference maps, and omit maps.

Servalcat - Cryo-EM Reciprocal-Space Refinement

Servalcat (Structure refinement and validation for crystallography and single particle analysis) performs reciprocal-space refinement and map calculation for cryo-EM SPA and crystallography.

Key advantage over real-space refinement: Produces properly weighted and sharpened Fo-Fc difference maps.

Installation

Servalcat is included in CCP4 (v9.0+). Alternatively:

pip install servalcat

Source: https://github.com/keitaroyam/servalcat

Documentation

• Main: https://servalcat.readthedocs.io/
• SPA refinement: https://servalcat.readthedocs.io/en/latest/spa.html
• Examples: https://servalcat.readthedocs.io/en/latest/spa_examples/index.html

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Basic Usage

servalcat <command> <args>

# Get help for any command
servalcat <command> -h

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Cryo-EM SPA Commands

1. Refinement (refine_spa_norefmac)

Reciprocal-space refinement against half-maps with automatic Fo-Fc map calculation.

# Basic refinement (run in a NEW directory!)
servalcat refine_spa_norefmac \
    --model model.pdb \
    --halfmaps half_map_1.mrc half_map_2.mrc \
    --resolution 3.0 \
    --ncycle 10

Important: Half-maps must be unsharpened and unweighted (e.g., from RELION Refine3D).

With Point Group Symmetry

# C3 symmetry - model is ASU only
servalcat refine_spa_norefmac \
    --model asu_model.pdb \
    --halfmaps half_map_1.mrc half_map_2.mrc \
    --resolution 2.5 \
    --pg C3 \
    --ncycle 10

Supported symmetry: Cn, Dn, T, O, I (follows RELION convention - center of box is origin).

With Helical Symmetry

servalcat refine_spa_norefmac \
    --model model.pdb \
    --halfmaps half_map_1.mrc half_map_2.mrc \
    --resolution 3.5 \
    --twist 179.4 --rise 2.4 \
    --ncycle 10

Common Options

Option	Description
--resolution	Resolution limit (use slightly higher than global FSC=0.143)
--ncycle N	Number of refinement cycles (default: 10)
--mask_for_fofc mask.mrc	Mask for Fo-Fc map calculation (doesn't affect refinement)
--ligand lig.cif	Restraint dictionary for ligands
--jellybody	Enable jelly-body refinement
--weight VALUE	Override automatic weight determination
--pixel_size VALUE	Override pixel size from header
--hydrogen {all,yes,no}	Hydrogen handling (default: all = regenerate)
--hout	Write hydrogens to output model
-o PREFIX	Output prefix (default: refined)
--cross_validation	Use half1 for refinement, half2 for validation

Output Files

File	Description
refined.pdb	Refined model (PDB format)
refined.mmcif	Refined model (mmCIF format)
refined_expanded.pdb	Symmetry-expanded model
refined_diffmap.mtz	Fo and Fo-Fc maps (auto-opens in Coot)
refined_diffmap_normalized_fofc.mrc	Normalized Fo-Fc map
refined_fsc.log	FSC curves (view with loggraph refined_fsc.log)

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2. Fo-Fc Map Calculation (fofc)

Calculate difference maps without refinement (use after refinement or for omit maps).

# Basic Fo-Fc calculation
servalcat fofc \
    --model refined.pdb \
    --halfmaps half_map_1.mrc half_map_2.mrc \
    --resolution 3.0 \
    --mask mask.mrc \
    -o diffmap

Omit Map Workflow

1. Refine complete model first
2. Remove atoms to omit (e.g., ligand) in Coot → save as model_omit.pdb
3. Calculate omit map:

servalcat fofc \
    --model model_omit.pdb \
    --halfmaps half_map_1.mrc half_map_2.mrc \
    --resolution 3.0 \
    --mask mask.mrc \
    -o omit_map

Hydrogen-Omit Map

servalcat fofc \
    --model refined.pdb \
    --halfmaps half_map_1.mrc half_map_2.mrc \
    --resolution 3.0 \
    --mask mask.mrc \
    --omit_proton \
    -o h_omit

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3. Map Trimming (trim)

Reduce map file sizes by removing empty regions.

servalcat trim \
    --maps postprocess.mrc half_map_1.mrc half_map_2.mrc \
    --mask mask.mrc \
    --padding 10

Options:

• --model model.pdb — Use model to define boundary (if no mask)
• --no_shift — Keep original origin (maps overlap with input)
• --noncubic — Don't enforce cubic box
• --noncentered — Don't center on original map

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Crystallographic Refinement

servalcat refine_xtal_norefmac \
    --model input.pdb \
    --hklin data.mtz \
    -s xray \
    --ncycle 10

Source options: xray, neutron, electron

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Utility Commands

Expand NCS/Symmetry

# Using Servalcat
servalcat util expand --model model.pdb

# Or using gemmi
gemmi convert --expand-ncs=new model.pdb model_expanded.pdb

Find Fo-Fc Peaks

servalcat util map_peaks \
    --map refined_diffmap_normalized_fofc.mrc \
    --model refined.pdb \
    --abs_level 4.0

Convert FSC JSON to CSV

servalcat util json2csv refined_fsc.json

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External Restraints (Refmac Keywords)

Servalcat supports Refmac-style keyword files for external restraints.

servalcat refine_spa_norefmac \
    --model model.pdb \
    --halfmaps half_1.mrc half_2.mrc \
    --resolution 3.0 \
    --keyword_file restraints.txt

Example Restraint File

# Distance restraint
external distance first chain A resi 50 atom CA \
                  second chain B resi 100 atom CA \
                  value 10.0 sigma 0.5

# Harmonic positional restraint
external harmonic first chain A resi 1:50 sigma 0.1

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Viewing Maps

In Coot

# Auto-generated script opens model + maps correctly
coot --script refined_coot.py

Note: Ignore "rmsd" sigma levels in Coot for SPA maps. Use raw map values directly (normalized within mask = true sigma).

In PyMOL

# MUST disable normalization before loading maps!
set normalize_ccp4_maps, off
load refined_diffmap_normalized_fofc.mrc
isomesh mesh_fofc, refined_diffmap_normalized_fofc, 4.0

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Tips

1. Always use unsharpened, unweighted half-maps from RELION Refine3D (not PostProcess)

2. Resolution: Set slightly higher than global FSC=0.143 (local resolution may be better)

3. Run in a new directory — Servalcat creates many files with fixed names

4. Check FSC curves with loggraph refined_fsc.log to assess overfitting

   • FSC_model should not exceed FSC_full_sqrt (indicates overfitting)

5. For omit maps: Refine complete model first, then remove atoms and recalculate maps

6. Sigma interpretation: Maps normalized within mask show true sigma levels; ignore Coot's "rmsd" display

7. Weight adjustment: If geometry is too loose/tight, adjust with --weight (larger = looser restraints)

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Workflow: Complete SPA Structure

# 1. Create working directory
mkdir refinement && cd refinement

# 2. Initial refinement
servalcat refine_spa_norefmac \
    --model ../initial_model.pdb \
    --halfmaps ../half_map_1.mrc ../half_map_2.mrc \
    --mask_for_fofc ../mask.mrc \
    --resolution 2.8 \
    --ncycle 10

# 3. Check results
coot --script refined_coot.py

# 4. If needed: edit model in Coot, save, re-refine
servalcat refine_spa_norefmac \
    --model refined_edited.pdb \
    --halfmaps ../half_map_1.mrc ../half_map_2.mrc \
    --mask_for_fofc ../mask.mrc \
    --resolution 2.8 \
    --ncycle 5 \
    -o refined_round2

# 5. Validate
molprobity.molprobity refined_expanded.pdb nqh=false

# 6. Calculate omit map for ligand (optional)
servalcat fofc \
    --model refined_no_ligand.pdb \
    --halfmaps ../half_map_1.mrc ../half_map_2.mrc \
    --mask ../mask.mrc \
    --resolution 2.8 \
    -o ligand_omit

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Citation

Cryo-EM SPA refinement:

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

Refmacat and restraint generation:

Yamashita, K., Wojdyr, M., Long, F., Nicholls, R. A., Murshudov, G. N. (2023) "GEMMI and Servalcat restrain REFMAC5" Acta Cryst. D79, 368-373