structures that can be solved to high resolution. It is therefore
timely to develop methods that can integrate the data-rich
MS-based approaches with cryo-EM.
2 Materials
The following software packages need to be downloaded and
installed:
1. PyMOL (https://pymol.org/2/).
2. Chimera (https://www.cgl.ucsf.edu/chimera/).
3. Integrative Modeling Platform (https://integrativemodeling.
org/).
4. IMPACT (http://impact.chem.ox.ac.uk/).
Webservers accessed as part of this protocol:
5. UniProt (https://www.uniprot.org/).
6. BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
7. T-Coffee (http://tcoffee.crg.cat/).
8. RCSB (http://www.rcsb.org/).
9. PDBe (http://www.ebi.ac.uk/pdbe/node/1).
10. EMDB (https://www.ebi.ac.uk/pdbe/emdb/).
All tutorial files can be downloaded from:
11. Github (https://github.com/andymlau/MIMB_Integrative_
Modelling).
3 Methods
All instructions below assume that tutorial files have been downloaded and that the required software packages have been installed
and tested for correct functionality.
3.1 Preparing Inputs
for IMP
1. The IMP platform can be used to generate models of protein
complexes, informed by experimental techniques which each
contribute modeling restraints. To use stoichiometry, crosslinking, and cryo-EM restraints, the following files are needed:
Files
initial_model.pdb—contains EM-map aligned model of the
protein complex.
sequences.fasta—contains full sequences of each subunit.
stoichiometry.txt—list of stoichiometric connectivities.
crosslinks.txt—list of experimental cross-links.
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Andy M. Lau and Argyris Politis
timely to develop methods that can integrate the data-rich
MS-based approaches with cryo-EM.
2 Materials
The following software packages need to be downloaded and
installed:
1. PyMOL (https://pymol.org/2/).
2. Chimera (https://www.cgl.ucsf.edu/chimera/).
3. Integrative Modeling Platform (https://integrativemodeling.
org/).
4. IMPACT (http://impact.chem.ox.ac.uk/).
Webservers accessed as part of this protocol:
5. UniProt (https://www.uniprot.org/).
6. BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
7. T-Coffee (http://tcoffee.crg.cat/).
8. RCSB (http://www.rcsb.org/).
9. PDBe (http://www.ebi.ac.uk/pdbe/node/1).
10. EMDB (https://www.ebi.ac.uk/pdbe/emdb/).
All tutorial files can be downloaded from:
11. Github (https://github.com/andymlau/MIMB_Integrative_
Modelling).
3 Methods
All instructions below assume that tutorial files have been downloaded and that the required software packages have been installed
and tested for correct functionality.
3.1 Preparing Inputs
for IMP
1. The IMP platform can be used to generate models of protein
complexes, informed by experimental techniques which each
contribute modeling restraints. To use stoichiometry, crosslinking, and cryo-EM restraints, the following files are needed:
Files
initial_model.pdb—contains EM-map aligned model of the
protein complex.
sequences.fasta—contains full sequences of each subunit.
stoichiometry.txt—list of stoichiometric connectivities.
crosslinks.txt—list of experimental cross-links.
224
Andy M. Lau and Argyris Politis