Rosetta 3.1 Release Manual

Documentation for membrane ab initio modeling application

Examples

See rosetta_source/test/integration/tests/membrane_abinitio for an example membrane ab initio run and input files.

Purpose and Algorithm

This protocol was developed to predict structures of helical membrane proteins with and without experimantal constraints. Briefly, the protocol is using transmembrane region prediction from OCTOPUS server to set initial membrane normal and membrane center vectors that are then used to define membrane-specific environment (hydrophobic core, interface, polar, and water layers). Using limited constraints based on residue-residue interactions obtained from protein structure databases or experiments, the protocol constrains helix-helix packing arrangements at particular positions.

Input Files

• Sample command line:

./bin/membrane_abinitio2.linuxgccrelease -in:file:native BRD4.pdb (or -in:file:fasta BRD4_.fasta) -in:file:spanfile BRD4.span -in:file:lipofile BRD4.lips4 -in:file:frag3 aaBRD4_03_05.200_v1_3 -in:file:frag9 aaBRD4_09_05.200_v1_3 -in:path:database ../rosetta_database -abinitio:membrane -membrane:no_interpolate_Mpair -membrane:Menv_penalties -nstruct 1 -score:find_neighbors_3dgrid -mute all

Membrane ab initio protocol specific input files (BRD4.span and BRD4.lips4 in the above example) generated using octopus2span.pl, run_lips.pl, and alignblast.pl scripts located in /rosetta_source/src/apps/public/membrane_abinitio/ . Note that blastpgp and nr database are necessary to run run_lips.pl script.

• BRD4.span - transmembrane topology prediction file generated using octopus2span.pl script as follows:

  octopus2span.pl <OCTOPUS topology file>
  
  example: <path to mini>/rosetta_source/src/apps/public/membrane_abinitio/octopus2span.pl BRD4.octopus
  

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

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

1st line is comment line. 2nd line shows number of predicted transmembrane helices (4 in the example above) and total number of residues (123 in the example above). 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).

• BRD4.lips4 - lipophilicity prediction file created using run_lips.pl script as follows:

  run_lips.pl <fasta file> <span file> <path to blastpgp> <path to nr database> <path to alignblast.pl script>
  
  example: ./rosetta_source/src/apps/public/membrane_abinitio/run_lips.pl BRD4.fasta BRD4.span ~/blast/bin/blastpgp /scratch/shared/genomes/nr ./rosetta_source/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
...

• Fragment files Generate structural fragments only using SAM secondary structure prediction file (*.rdb) - jufo and psipred predict transmembrane helical regions poorly.

Command line arguments

You can run membrane ab initio modeling with the following flags:

-abinitio:membrane
-in:file:native inputs/BRD4.pdb
-in:file:frag3 inputs/aaBRD4_03_05.200_v1_3
-in:file:frag9 inputs/aaBRD4_09_05.200_v1_3
-in:file:spanfile inputs/BRD4.span
-in:file:lipofile inputs/BRD4.lips4
-membrane:no_interpolate_Mpair
-membrane:Menv_penalties
-score:find_neighbors_3dgrid
-out:nstruct 1
-out:file:silent BRD4_silent.out
-mute all

For more information about membrane ab initio modeling flags, please check the @ref options "options list"

Please note:

-membrane:no_interpolate_Mpair
This flag will switch off the interpolation between the two layers for the pair-term to mimic the old code

-membrane:Menv_penalties
This flag will switch on the following penalties:

    * no non-helical secondary structure fragments in predicted transmembrane helical regions in the hydrophobic layer of the membrane.
    * no N- and C- termini residues of predicted transmembrane helical regions in the hydrophobic layer of the membrane.
    * no transmembrane helices with orientation >45 degrees relative to the membrane normal vector.

-smooth_move_frac flag is not being used and will be removed in the next release.

@section result Result Interpretation
During membrane ab initio run, protocol generates pdb files and an energy file.
Membrane ab initio application specific score outputs in the energy file are:
@verbatim
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

References

• Barth P, Wallner B, Baker D. (2009 Feb 3) Prediction of membrane protein structures with complex topologies using limited constraints. Proc Natl Acad Sci U S A. 106(5):1409-14.
• Barth P, Schonbrun J, Baker D. (2007 Oct 2) Toward high-resolution prediction and design of transmembrane helical protein structures. Proc Natl Acad Sci U S A. 104(40):15682-7.
• Yarov-Yarovoy V, Schonbrun J, Baker D. (2006 Mar 1) Multipass membrane protein structure prediction using Rosetta. Proteins. 62(4):1010-25.

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