Fusing C-terminal or N-terminal segments onto proteins using Remodel

[Anonymous]

Hi I am trying to fuse a structure onto a designed protein structure at the C-terminus using the following command:

remodel.linuxgccrelease -s model3.pdb -remodel:RemodelLoopMover:bypass_closure -remodel:domainFusion:insert_segment_from_pdb model4.pdb -remodel:blueprint model3.blueprint -use_input_sc -num_trajectory 3 -save_top 1 -database <PATH> -overwrite -out:file:scorefile model3.sc

model3.pdb covers the range: 1-393

model4 covers the range: 290 – 393

Where the blueprint has the following general format:

---

<All preceding residues unchanged>

280 R .

281 D .

282 R .

283 R .

284 T .

285 E .

286 E L PIKAA E

287 E L PIKAA E

288 N L PIKAA N

289 L L PIKAA L

0 x I NATRO

0 x I NATRO

0 x I NATRO

0 x I NATRO

<The remaining residues treated as insertion and followed by EOF>

---

However when it is run the following error occurs:

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protocols.forge.remodel.RemodelWorkingSet: segment contains insertion, skip normal SegmentRebuild instructions, use SegmentInsert instructions instead

protocols.forge.remodel.RemodelWorkingSet: beforeInsert: LLLL

protocols.forge.remodel.RemodelWorkingSet: afterInsert:

protocols.forge.remodel.RemodelWorkingSet: insert_SS_string: LLLL^

core.conformation.Residue: [ WARNING ] missing an atom: 1  H   that depends on a nonexistent polymer connection!

core.conformation.Residue:  –> generating it using idealized coordinates.

ERROR: seqpos >= 1

ERROR:: Exit from: src/core/conformation/Conformation.hh line: 379

/mnt/Programs/Rosetta/rosetta_bin_linux_2015.12.57698_bundle/main/source/build/src/release/linux/2.6/64/x86/gcc/4.4/default/libutility.so(print_backtrace()+0x23) [0x7f3b935fc093]

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I have looked for this error and it seems to pop us in the protein design segment, but I am not quite sure where or how to correct it. Any thoughts would be welcome.

[Anonymous]

Hi I have figured part of the work-around for the above problems. Now it crashes in the process of manual design. If the inserted region is on the n-terminus of the full model it passes through the first stages of the loop modeling before crashing. 

 

remodel.linuxgccrelease -s model3.pdb  -remodel:domainFusion:insert_segment_from_pdb model4-nterm.pdb -remodel:blueprint model3-nterm.blueprint -use_input_sc -save_top 1 -overwrite -out:file:scorefile model3-nterm.sc

model3.pdb covers the range: 1-393

model4-nterm covers the range: 1-293

Where the blueprint has the following general format:

---

 

<All preceding residues inserted>

0 x I NATRO

0 x I NATRO

0 x I NATRO

0 x I NATRO

294 E L PIKAA E

295 P L PIKAA P

296 H L PIKAA H

297 H L PIKAA H

< All succeeding residues unchanged and followed by EOF>

---

 

Incomplete: Ends after initial moving and scoring

---

 

protocols.forge.remodel.RemodelLoopMover: ** randomize_stage

protocols.forge.remodel.RemodelLoopMover: * closure_attempt 1

protocols.forge.remodel.RemodelLoopMover: ** simultaneous_stage

protocols.forge.remodel.RemodelLoopMover:    n_loops = 1

protocols.forge.remodel.RemodelLoopMover:

---

 Scores                       Weight   Raw Score Wghtd.Score

---

 vdw                          1.000       5.369       5.369

 hbond_sr_bb                  1.000    -136.160    -136.160

 hbond_lr_bb                  1.000      -3.063      -3.063

 rama                         0.100     -24.283      -2.428

 rg                           1.000      39.446      39.446

---

 Total weighted score:                      -96.836

 

protocols.moves.MonteCarlo: MC: 2  -66.7454  -96.8365  -66.7454  -96.8365  0  0  0  rejected

protocols.moves.TrialCounter:       simul_frag trials=  23760;  accepts= 0.1666;  energy_drop/trial=  -0.07959

protocols.forge.remodel.RemodelLoopMover: ** independent_stage

protocols.forge.remodel.RemodelLoopMover:    n_loops = 1

protocols.forge.remodel.RemodelLoopMover:

---

 Scores                       Weight   Raw Score Wghtd.Score

---

 vdw                          1.000       4.618       4.618

 hbond_sr_bb                  1.000    -145.384    -145.384

 hbond_lr_bb                  1.000      -2.082      -2.082

 rama                         0.100     -41.183      -4.118

 rg                           1.000      41.436      41.436

---

 Total weighted score:                     -105.531

 

protocols.moves.MonteCarlo: MC: 2  -86.9267  -105.531  -86.9267  -105.531  0  0  0  rejected

protocols.moves.TrialCounter:             frag trials=  95040;  accepts= 0.1638;  energy_drop/trial=   0.00010

protocols.forge.remodel.RemodelLoopMover: ** boost_closure_stage

protocols.forge.remodel.RemodelLoopMover:    n_loops = 0

protocols.forge.remodel.RemodelLoopMover:    no loops to work on with boost_closure. returning.

protocols.forge.remodel.RemodelLoopMover: * 1 / 1   closed / attempts

core.scoring.ScoreFunctionFactory: SCOREFUNCTION: talaris2013

core.pack.task: Packer task: initialize from command line()

core.pack.interaction_graph.interaction_graph_factory: Instantiating DensePDInteractionGraph

core.pack.pack_rotamers: built 0 rotamers at 0 positions.

core.pack.pack_rotamers: IG: 472 bytes

core.scoring.ScoreFunctionFactory: SCOREFUNCTION: talaris2013

core.pack.task: Packer task: initialize from command line()

core.pack.interaction_graph.interaction_graph_factory: Instantiating DensePDInteractionGraph

core.pack.pack_rotamers: built 0 rotamers at 0 positions.

core.pack.pack_rotamers: IG: 472 bytes

protocols.forge.remodel.RemodelMover: centroid_build(): variable length build succeeded.

protocols.forge.remodel.RemodelMover: apply(): calling RemodelDesignMover apply function.

protocols.forge.remodel.RemodelDesignMover: Design Mover state: stage

protocols.forge.remodel.RemodelDesignMover: MODE 4: Manual DESIGN REMODEL

core.pack.task: Packer task: initialize from command line()

core.pack.task.ResfileReader: On line 3, the pose does not have residue with chain= , PDBnum=1.

ERROR:: Exit from: src/core/pack/task/ResfileReader.cc line: 1520

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