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

Author: Rebecca F. Alford (rfalford12@gmail.com)
Corresponding PI: Jeffrey J. Gray (jgray@jhu.edu)
Last Updated: January 2015
Rosetta Revision #58069

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:

• Alford RF, Koehler Leman J, Weitzner BD, Duran A, Elazar A, Tiley D, Gray JJ (2015) An integrated framework advancing membrane protein modeling and design PLoS ONE (in preparation)

## Executable/Script

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:

Rosetta/main/source/bin/rosetta_scripts.linuxgccrelease


## Generating Inputs

Two inputs are required for the membrane relax application:

1. PDB for the protein structure of interest

2. 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:

1. 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).

2. 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:

• 3pxo_tr.span: Spanfile containing predicted trans-membrane spans

## Steps of the protocol

Here, we describe the steps required to run the MP_Relax protocol. As an example, all steps use the PDB 3pxo:

1. 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

2. Recommended # of Decoys

• For demo run: 1
• For production runs: 1000
3. Command line:

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.

## Example Outputs

The following outputs will be generated from the relax protocol. A version of these outputs are also provided in the example_outputs/ directory:

• 3pxo_tr_0001.pdb : Output refined model of 3pxo
• relax_scores_3pxo.sc : Rosetta scores (including membrane scores) for refinement run

## References

1. 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.

2. 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.

3. 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.