Overview

This tutorial will show you how to fit biomolecule atomic structures into cryo-EM maps using molecular dynamics simulation with PHENIX commandline

For GUI execution, please see the cryo_fit_gui_tutorial.

Theoretical explanation of cryo_fit is here.

For installation of cryo_fit, please see the installation notes for cryo_fit.

Input files

<initial_model> and <target_map>

Initial Model

Available format: .cif and .pdb

The initial model is a guide or template structure (CIF/mmCIF/pdb) that is close to a target cryo EM map structurally.

A user can use either map_to_model or UCSF chimera (Tools -> Volume Data -> Fit in Map) to prepare the initial model.

Target Map

Available format: .ccp4 and .map (MRC style in binary file) and .sit (Situs style in text file)

Running the program

• phenix.cryo_fit <initial_model> <target_map>

• For example, phenix.cryo_fit model.pdb map.ccp4

Running the tutorial

• Use files in <user phenix>/modules/cryo_fit/tutorial_input_files

• For example, phenix.cryo_fit GTPase_activation_center_tutorial.pdb GTPase_activation_center_tutorial_gaussian_1p5.mrc

Output

• "Output" folder has all useful output files
• By default, cryo_fit reports the 3 highest cc files:

• cryo_fitted_chain_recovered_cleaned_for_real_space_refine_molprobity.pdb
• extracted_<x>_ps.pdb
• extracted_<y>_ps.pdb

• A final cryo_fitted structure (The highest cc file):

• cryo_fitted_chain_recovered_cleaned_for_real_space_refine_molprobity.pdb

• <x>.gro file is for vmd visualization.
• Geometry extracted files at certain (x) pico second during MD simualtion:

• extracted_<x>_ps.pdb

• The user provided file:

• user_provided.pdb (when cryo_fit doesn't find higher cc than a user provided.pdb, this file exists in output folder)

• Correlation coefficients (CC) between cryo_fitted structures and cryo-EM maps:

• cc_record

• If a user wants the highest cc file, then use either

• cryo_fitted_chain_recovered_cleaned_for_real_space_refine_molprobity.pdb
• or
• user_provided.pdb

• If a user wants other files among the highest cc valued files, then use

• extracted_<x>_ps.pdb

Example

Limitation

gromacs4.5.5 seems to not handle H2O (water) heteroatom. cryo_fit will remove water molecules (if any) from the input .cif/.pdb and fit to cryo electron microscopy map.

cryo_fit doesn't handle non-canonical "residue"s such as 7C4, BMA, GDP, ILX, NAG, SEP, TRX. The cryo_fit will simply exclude those residues and report what are excluded.

How to generate and record movie?

Generate_record_movie_by_cryo_fit1

Options

All options will be used as default if unspecified. Gromacs expert users are welcome to customize those options if they wish.

List of most useful options

Option	Default value	Description of inputs and uses
emweight_multiply_by	8	Multiply by this number to the number of atoms for weight for cryo-EM map bias. For example, emweight = (number of atoms in gro file) x (emweight_multiply_by which is 8) The higher the weight, the stronger bias toward EM map rather than MD force field and stereochemistry preserving constraints. If user's map has a better resolution, higher value of emweight_multiply_by is recommended since map has much information. If user's map has have a worse resolution, lower value of emweight_multiply_by is recommended for more likely geometry. If CC (correlation coefficient) needs to be improved faster, higher number of emweight_multiply_by is recommended.
nproc	max cores	Specify number of cores for minimization and cryo_fit. If it is not specified, or max is chosen, the cryo_fit will try to use most cores automatically (up to 16)
number_of_steps_for_cr yo_fit	None	This is the initial number of steps for cryo_fit. Eventually, cryo_fit will increase it depending on molecule size and cc trend. For tutorial files, this will be 70,000
number_of_steps_for_mi nimization	None	Specify number of steps for minimization. If this is left blank, cryo_fit will estimate it depending on molecule size.number of steps for cryo_fit. Enough minimization will prevent "blow-up" during MD simulation later.