Can Fold-and-dock protocol applied to membrane proteins

[Anonymous]

Dear all,

I have a membrane protein which is a dimer. I want to fold the monomer and to assemble it to a dimer.
Can Rosetta3.2 or 3.2.1 do it?
If yes, please give some advice on option setup.

Thank you!

-Justin

[Anonymous]

No one knows the solution?

I have tried the following options:
-database /home/clzhang/rosetta3.2/rosetta_database

-in:file:fasta
-in:file:frag3
-in:file:frag9
-in:file:spanfile
-in:file:lipofile
-symmetry_definition symm_def_dimer.dat
-symmetry:initialize_rigid_body_dofs

-abinitio:membrane
-membrane:no_interpolate_Mpair
-membrane:Menv_penalties

But I got complains like: “core.scoring.SymmetricScoreFunction: Warning!!! Using a symmetric score function on a non-symmetric pose”

Does anyone knows what the problem is?
Or, currently, symmetry file cannot be recognized by membrane_denovo executable?

-Justin

[Anonymous]

Justin, how did you get on with this? any luck?

Chris

[Anonymous]

The command above is completely wrong and took me down a blind alley.

My current command looks like this….

/home/chxcja/rosetta3.3_bundles/rosetta_source/bin/minirosetta.linuxgccrelease
-run:protocol broker
-broker:setup setup_init.tpb

-in:file:fasta /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/input/eliza.FASTA
-in:file:frag3 /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/input/aat000_03_05.200_v1_3
-in:file:frag9 /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/input/aat000_09_05.200_v1_3
-in:file:spanfile /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/input/eliza.span
-in:file:lipofile /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/input/eliz.lips4
-symmetry:symmetry_definition /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/input/elizaC4.symm
-symmetry:initialize_rigid_body_dofs
-database /home/chxcja/rosetta3.3_bundles/rosetta_database/

-abinitio:membrane
-membrane:no_interpolate_Mpair -membrane:Menv_penalties -membrane:normal_cycles 100 -membrane:normal_mag 5 -membrane:center_mag 1

-fold_and_dock:rigid_body_cycles 1
-fold_and_dock:rigid_body_frequency 5
-fold_and_dock:rotate_anchor_to_x
-run:reinitialize_mover_for_each_job
-score:weights score13_env_hb
-abinitio:recover_low_in_stages 0
-abinitio:rg_reweight 0.001
-abinitio:use_filters false
-packing:ex1
-packing:ex1:level 1
-packing:ex2
-packing:ex2:level 1
-packing:extrachi_cutoff 0

-evaluation:symmetric_rmsd
-nstruct 20
-out:file:silent_struct_type binary
-relax:quick
-relax:jump_move

-out:path:pdb /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock/pdb
-out:pdb
-out:file:silent silent.out
-no_prof_info_in_silentout
-mute core.io.database
-out:level 500
-out:file:scorefile score.sc

However I’m not sure if this is using any of the membrane settings – i get something that looks vaguely like a pore type structure but the helices are at weird angles (Ie greater than the Menv penalties should allow I think).

I’ll keep people posted on my progress but if anyone has any ideas I’d really like to hear them.

Thanks

Chris

[Anonymous]

EDITED NOTE: The following command is useless. Do not waste your time with it. I have left it here for the record but please don’t run this….

---

I run fold and dock with the following command (see below) but when I run

/home/chxcja/rosetta3.3_bundles/rosetta_source/bin/score.linuxgccrelease -database ~/rosetta3.3_bundles/rosetta_database/ -in:file:silent default-c4membrane.out -in::file::fullatom -out:output

to get a pdb file of the models the pdbs only contain 1 chain. How can I get the pdb of the complex (in this case a C4 symmetric structure).

Thanks for your help

Chris

***********************************************
/home/chxcja/rosetta3.3_bundles/rosetta_source/bin/membrane_abinitio2.linuxgccrelease
-run:protocol broker
-broker:setup setup_init.tpb

-out:file:scorefile score-membrane.sc
-in:path:database /home/chxcja/rosetta3.3_bundles/rosetta_database/

-file:frag3 aat000_03_05.200_v1_3
-file:frag9 aat000_09_05.200_v1_3

-symmetry:symmetry_definition elizaC4.symm
-out:file:silent default-c4membrane.out
-out:file:silent_struct_type binary
-relax:fast
-relax:jump_move
-symmetry:initialize_rigid_body_dofs

-in:file:fasta eliza.FASTA
-in:file:spanfile eliza.span
-in:file:lipofile eliz.lips4

-abinitio:membrane
-score:find_neighbors_3dgrid
-membrane:no_interpolate_Mpair
-membrane:Menv_penalties
-membrane:normal_cycles 1000
-membrane:normal_mag 5
-membrane:center_mag 1
-nstruct 5

-fold_and_dock:rigid_body_cycles 1
-fold_and_dock:rigid_body_frequency 5
-fold_and_dock:rotate_anchor_to_x

-rg_reweight 0.001
-rigid_body_cycles 1
-abinitio::recover_low_in_stages 0
-rigid_body_frequency 5
-rigid_body_disable_mc
-run:reinitialize_mover_for_each_job
> rosetta.log &

[Anonymous]

These are the flags that I tried to fold M2 channel, and got a GDTMM 0.70 to the native structure as a tetramer. Hope this helpful –

-run:protocol broker
-broker:setup setup_init.tpb
-database minirosetta_database
-nstruct 100
-out:file:silent_struct_type binary

-in:file:fasta 2kqtA.fasta
-file:frag3 2kqtA.fasta.frags.3mers
-file:frag9 2kqtA.fasta.frags.9mers

-rg_reweight 0.01
-run:reinitialize_mover_for_each_job

-score:find_neighbors_3dgrid

-symmetry:symmetry_definition C4.symm
-symmetry:initialize_rigid_body_dofs
-fold_and_dock::rotate_anchor_to_x

-relax:fast
-relax:jump_move

-abinitio:membrane
-membrane:fixed_membrane
-membrane:no_interpolate_Mpair
-membrane:Menv_penalties

-in:file:spanfile 2kqtA.span
-in:file:lipofile 2kqtA.lips4
-stage2_patch score_membrane_s2.wts_patch
-stage3a_patch score_membrane_s3a.wts_patch
-stage3b_patch score_membrane_s3b.wts_patch
-stage4_patch score_membrane_s4.wts_patch

-membrane:Membed_init
-relax:membrane
-score:weights membrane_highres.wts

-increase_cycles 4.3
-evaluation::gdtmm

[Anonymous]

Thanks Wangyr, following your suggestions it was clear that there were a few things missing from my commands.

Adding them however goves me an odd result where the actual chains are not in contact with one another.

Can anyone suggest what I might be doing wrong?

Thanks

Chris

Binary output file
http://dl.dropbox.com/u/1184313/SIT-membrane5.out

Commands
############
/home/chxcja/rosetta3.3_bundles/rosetta_source/bin/minirosetta.linuxgccrelease
-run:protocol broker
-broker:setup setup_init.tpb

-in:file:fasta /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/eliza.FASTA
-in:file:frag3 /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/aat000_03_05.200_v1_3
-in:file:frag9 /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/aat000_09_05.200_v1_3
-in:file:spanfile /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/eliza.span
-in:file:lipofile /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/eliz.lips4
-symmetry:symmetry_definition /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock3/input/elizaC4.symm
-symmetry:initialize_rigid_body_dofs
-database /home/chxcja/rosetta3.3_bundles/rosetta_database/
-nstruct 20

-abinitio:membrane
-membrane:no_interpolate_Mpair
-membrane:Menv_penalties
-membrane:normal_cycles 200
-membrane:normal_mag 5
-membrane:center_mag 1
-membrane:Membed_init
-abinitio:recover_low_in_stages 0
-abinitio:rg_reweight 0.001

-fold_and_dock:rigid_body_cycles 1
-fold_and_dock:rigid_body_frequency 5
-fold_and_dock:rotate_anchor_to_x
-run:reinitialize_mover_for_each_job
-score:weights score13_env_hb

-packing:ex1
-packing:ex1:level 1
-packing:ex2
-packing:ex2:level 1
-packing:extrachi_cutoff 0
-score:find_neighbors_3dgrid

-evaluation:symmetric_rmsd
-relax:quick
-relax:jump_move

-abinitio:stage2_patch /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/score_membrane_s2.wts_patch
-abinitio:stage3a_patch /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/score_membrane_s3a.wts_patch
-abinitio:stage3b_patch /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/score_membrane_s3b.wts_patch
-abinitio:stage4_patch /home/chxcja/rosetta3.3_bundles/SIT-membrane-fold-dock5/input/score_membrane_s4.wts_patch

-out:pdb
-out:file:silent_struct_type binary
-out:file:silent SIT-membrane5.out
-no_prof_info_in_silentout
-mute core.io.database
-out:file:scorefile score.sc

-relax:membrane
-score:weights membrane_highres.wts
-increase_cycles 4.3

[Anonymous]

Folding membrane protein in Rosetta is always really tricky. Is this protein a channel that has charged residues in the core? If that’s the case, there are a lot parameterization need to benchmark in energy function for this kind of protein, which no one has done that yet.

Could you run a positive control to see whether this is a target protein problem or a setup problem? Using the M2 channel fasta I posted previously will be an easy and quick test.

Also, it seems like the protein you are working on is really big. Please take a look at Rhiju’s fold’n dock PNAS paper to see what’s the size limitation and how much sampling you need to have.

Best,
Ray

[Anonymous]

I don’t think that membrane_denovo can handle symmetry, although I don’t know for sure. The error is consistent with this hypothesis.

[Anonymous]

What does it do if you run it without the membrane stuff? It may be that the membrane scorefunction changes make Rosetta less likely to shove stuff together.

[Author]

Posts