The scripts and input files that accompany this demo can be found in the
demos/public directory of the Rosetta weekly releases.
KEYWORDS: MEMBRANES STRUCTURE_PREDICTION
This document was created on August 5, 2011 by:
This protocol was developed to predict helical membrane protein structures.
This protocol will only generate low-resolution centroid models. The protocol is using transmembrane region predictions from OCTOPUS server (http://octopus.cbr.su.se/) to set initial membrane normal and membrane center vectors and define membrane-specific environment (hydrophobic core, interface, polar, and water layers). For multispan helical membrane proteins the protocol starts from embedding only two randomly selected adjacent transmembrane helical regions and then continues folding by inserting one of adjacent helices until all transmembrane helices will be embedded into the membrane.
Generate structure fragments:
make_fragments.pl -verbose -id BRD4 BRD4_.fasta -nojufo -nopsipred
Documentation at http://www.rosettacommons.org/manuals/archive/rosetta3.3_user_guide/file_fragments.html. Note: use only SAM secondary structure prediction file (*.rdb). jufo and psipred predict transmembrane helical regions poorly.
Generate transmembrane regions (OCTOPUS) file:
Input OCTOPUS topology file is generated at http://octopus.cbr.su.se/ using protein sequence as input.
Sample OCTOPUS topology file:
##############################################################################
OCTOPUS result file
Generated from http://octopus.cbr.su.se/ at 2008-09-18 21:06:32
Total request time: 6.69 seconds.
##############################################################################
Sequence name: BRD4
Sequence length: 123 aa.
Sequence:
PIYWARYADWLFTTPLLLLDLALLVDADQGTILALVGADGIMIGTGLVGALTKVYSYRFV
WWAISTAAMLYILYVLFFGFTSKAESMRPEVASTFKVLRNVTVVLWSAYPVVWLIGSEGA
GIV
OCTOPUS predicted topology:
oooooMMMMMMMMMMMMMMMMMMMMMiiiiMMMMMMMMMMMMMMMMMMMMMooooooMMM
MMMMMMMMMMMMMMMMMMiiiiiiiiiiiiiiiiiiiiiMMMMMMMMMMMMMMMMMMMMM
ooo
Convert OCTOPUS file to .span file format:
BRD4.span: transmembrane topology prediction file generated using
octopus2span.pl script as follows:
octopus2span.pl <OCTOPUS topology file>
Example:
<path to mini>/mini/src/apps/public/membrane_abinitio/octopus2span.pl BRD4.octopus
Sample .span file:
TM region prediction for BRD4 predicted using OCTOPUS
4 123
antiparallel
n2c
6 26 6 26
31 51 31 51
58 78 58 78
97 117 97 117
Format for .span files:
1st line is comment line.
2nd line shows number of predicted transmembrane helices (4 in the command lines example below) and total number of residues (123 in the example below).
3rd line shows predicted topology of transmembrane helices in the membrane (currently only antiparallel topology is implemented).
4th line and all lines below show start and end residue numbers of each of the predicted transmembrane helices (current format repeats these numbers twice).
Generate .lips4 file.
BRD4.lips4: lipophilicity prediction file created using run_lips.pl
script as follows (note that blastpgp and nr database are necessary to run
run_lips.pl script:
run_lips.pl <fasta file> <span file> <path to blastpgp> <path to nr database> <path to alignblast.pl script>
Example:
<path to mini>/mini/src/apps/public/membrane_abinitio/run_lips.pl BRD4.fasta BRD4.span /work/bjornw/Apps/blast/bin/blastpgp /scratch/shared/genomes/nr ~bjornw/mini/src/apps/public/membrane_abinitio/alignblast.pl
Sample lips4 file:
Lipid exposed data: resnum mean-lipo lipophil entropy
6 -1.000 3.004 1.211
9 -1.000 2.268 2.137
10 -1.000 4.862 1.095
13 -1.000 1.304 1.552
16 -1.000 3.328 2.025
...
Run membrane ab initio application with the following flags (see an example in scripts/membrane-abinitio.cmd).:
./bin/membrane_abinitio2.linuxgccrelease
-in:file:native BRD4.pdb Native structure (optional)
(or -in:file:fasta BRD4_.fasta) Protein sequence in fasta format (required if native structure is not provided)
-in:file:spanfile BRD4.span Octopus transmembrane prediction (see above)
-in:file:lipofile BRD4.lips4 Lipophilicity prediction (see above)
-in:file:frag3 aaBRD4_03_05.200_v1_3 3-residue fragments
-in:file:frag9 aaBRD4_09_05.200_v1_3 9-residue fragments
-in:path:database ~minirosetta_database Path to rosetta database
-abinitio:membrane Membrane ab initio application
-score:find_neighbors_3dgrid Use a 3D lookup table for residue neighbors calculations
-membrane:no_interpolate_Mpair Switch off the interpolation between the two layers for the Mpair term
-membrane:Menv_penalties Switch on the following penalties:
1. no non-helical secondary structure fragments in predicted transmembrane helical regions in the hydrophobic layer of the membrane.
2. no N- and C- termini residues of predicted transmembrane helical regions in the hydrophobic layer of the membrane.
3. no transmembrane helices with orientation >45 degrees relative to the membrane normal vector.
-nstruct 1 Number of output structures
You have a choice to use either Monte Carlo (default) or discrete search of membrane normal and membrane center.
Optional settings used for Monte Carlo based membrane normal and center search protocol:
-membrane:normal_cycles (default=100) Total number of membrane normal cycles
-membrane:normal_mag (default=5) Magnitude of membrane normal angle search step size (degrees)
-membrane:center_mag (default=1) Magnitude of membrane center search step size (Angstroms)
Tip: to speedup Monte Carlo based membrane normal and center search use the following settings:
-membrane:normal_cycles 40
-membrane:normal_mag 15
-membrane:center_mag 2
Optional settings for alternative discrete search of membrane normal and membrane center:
-membrane:center_search (default= false) - perform membrane center search within "center_max_delta" deviation (see below).
-membrane:normal_search (default= false) - perform membrane normal search with normal_start_angle, normal_delta_angle, and normal_max_angle values (see below).
-membrane:center_max_delta (default= 5 A) - magnitude of maximum membrane width deviation during membrane center search (Angstroms).
-membrane:normal_start_angle (default= 10 degrees) - magnitude of starting angle during membrane normal search (degrees).
-membrane:normal_delta_angle (default= 10 degrees) - magnitude of angle deviation during membrane normal search (degrees).
-membrane:normal_max_angle (default= 40 degrees) - magnitude of maximum angle deviation during membrane normal search (degrees).
To run the example script, set your ROSETTA3 environment variable to your directory and type,
$> $ROSETTA3/bin/membrane_abinitio2.default.linuxgccrelease @scripts/flagsExpected Outputs
Convert output silent file into pdb file using score application as follows (see an example in scripts/membrane-centroid-score.cmd):
/Users/vyarovyarovoy/mini/bin/score.macosgccrelease \
-in:file:native rosetta_inputs/BRD4.pdb \
-in:file:centroid_rosetta_inputs/ \
-in:file:silent BRD4_silent.out \
-in:file:silent_struct_type binary \
-in:file:spanfile rosetta_inputs/BRD4.span \
-in:file:lipofile rosetta_inputs/BRD4.lips4 \
-membrane:no_interpolate_Mpair \
-membrane:Menv_penalties \
-score:find_neighbors_3dgrid \
-score:weights score_membrane.wts \
-out:nstruct 1 \
-database /Users/vyarovyarovoy/minirosetta_database \
-out:output \
-nstruct 1
To run this example, type
$> $ROSETTA3/bin/score_jd2.macosclangrelease @scripts/score_flagsMembrane ab initio application specific score outputs in the output score file are:
Mpair membrane pairwise residue interaction energy
Menv membrane residue environment energy
Mcbeta membrane residue centroid density energy
Mlipo membrane residue lipophilicity energy
Menv_hbond membrane non-helical secondary structure in the hydrophobic layer penalty
Menv_termini membrane N- and C-temini residue in the hydrophobic layer penalty
Menv_tm_proj transmembrane helix projection penalty
Generate at least 10,000 models and then use a clustering application to identify most frequently sampled conformations. In general case, at least one of top 5-10 clusters will have models with the lowest rmsd to the native structure.
Good luck :)!