The scripts and input files that accompany this demo can be found in the
demos/protocol_captures directory of the Rosetta weekly releases.
This is a protocol capture, and represents the protocol at a fixed point in time. It may not work with the current version of Rosetta.
KEYWORDS: MEMBRANES STRUCTURE_PREDICTION
High-resolution refinement is key for advancing low resolution structures from x-ray crystallography to atomic level detail. For membrane proteins, this method can also reveal an ensemble of possible membrane embeddings: the position and orientation of the biomolecule with respect to the membrane bilayer.
The membrane relax application combines the Rosetta FastRelax algorithm with the all atom energy function for membrane proteins and a gradient-based technique for optimizing the membrane embedding. First, a series of small backbone moves, rotamer trials, and minimization are used to refine the protein structure. In addition, the membrane position is optimizied by minimizing the "jump" or connecting relating the MEM residue to the biomolecule.
Publication describing the method:
The membrane framework relax application is implemented in Rosetta script. This script, called membrane_relax.xml is included in the main directory of this protocol capture.
It can be run with the following executable:
Two inputs are required for the membrane relax application:
PDB for the protein structure of interest
Span file describing the location of trans-membrane spans
Steps for generating these inputs are found below. A set of example inputs can also be found in example_inputs/. Here, metarhodopsin II (PDB ID: 3pxo) is used as an example:
PDB File: Generate a PDB file where the membrane protein structure is transformed into PDB coordinates (z-axis is membrane normal). This can be done either by downloading the transformed PDB directly from the PDBTM website (http://pdbtm.enzim.hu/) or by downloading a PDB file from the PDB and running it through the PPM server (http://opm.phar.umich.edu/server.php).
Span File: Generate a spanfile from the PDB structure using the spanfile_from_pdb application described in the MP_spanfile-from-pdb protocol capture in Rosetta/demos/protocol_captures/2014. An example commandline using 3pxo is also provided here:
Rosetta/main/source/bin/spanfile_from_pdb.linuxgccrelease -database /path/to/db -in:file:s example_inputs/3pxo_tr.pdb
For this example, this command will produce 1 output file:
Here, we describe the steps required to run the MP_Relax protocol. As an example, all steps use the PDB 3pxo:
Required Options: Options (flags) needed to run this application. A file with these flags, relax_flags, is also provided for 3pxo in this demo:
flags descriptions -------------------------------------------------------------------------------------------------- -parser:protocol membrane_relax.xml Use the membrane relax protocol Rosetta script -in:file:s Input PDB Structure: PDB file for protein structure -membrane_new:setup:spanfiles Spanfile describing trans-membrane spans of the starting structure -membrane_new:scoring:hbond Turn on membrane depth-dependent hydrogen bonding weight -relax:fast Use the FastRelax mode of Rosetta Relax (uses 5-8 repeat cycles) -relax:jump_move true Allow the MEM and other jumps to move during refinement -nstruct Number of structures to generate -packing:pack_missing_sidechains 0 Wait to pack until the membrane mode is turned on -out:pdb Output all PDB structures of refined models -out:file:scorefile Specify destination for score file
Recommended # of Decoys
To run this application, use the following command line:
Rosetta/main/source/bin/rosetta_scripts.linuxgccrelease -database /path/to/db @relax_flags
Note on timing: Refinement in Rosetta is a time consuming application. Depending on avaialble computing power and size of the protein, refinement of an individual decoy can take between 10-15min for ~200 residues and between 0.5-1.0hrs for proteins > 200 residues.
The following outputs will be generated from the relax protocol. A version of these outputs are also provided in the example_outputs/ directory:
Tyka MD, Keedy DA, Andre I, DiMaio F, Song Y, et al. (2011) Alternate states of proteins revealed by detailed energy landscape mapping. J Mol Biol.
Barth P, Schonbrun J, Baker D (2007) Toward high-resolution prediction and design of transmembrane helical protein structures. Proc Natl Acad Sci 104: 15682–15687.
Fleishman SJ, Leaver-Fay A, Corn JE, Strauch E-M, Khare SD, et al. (2011) RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite. PLoS ONE 6: e20161.