problems with terminal caps

[Anonymous]

I am having troubles with terminal caps in FlexPepDocking in Rosetta 3.5. In the input PDB files the receptor and peptide contain ACE and NME at the N- and C-terminus, respectively. Rosetta does not recognize the modified terminal residues as N_acetylated and C_methylamidated. I have carefully modified the ACE/NME atom names to correspond to the Rosetta naming scheme in the corresponding residue patch files. So far, I haven’t gotten any further than the prepacking phase of FlexPepDocking, because Rosetta gives me a warning, that it can not identify the residue type of the terminal residues and has thus discarded several atoms:


core.io.pdb.file_data: [ WARNING ] discarding 6 atoms at position 1 in file potra_pep_dock.pdb. Best match rsd_type: THR_p:NtermProteinFull
core.io.pdb.file_data: [ WARNING ] discarding 6 atoms at position 231 in file potra_pep_dock.pdb. Best match rsd_type: SER_p:CtermProteinFull
core.io.pdb.file_data: [ WARNING ] discarding 6 atoms at position 232 in file potra_pep_dock.pdb. Best match rsd_type: ILE_p:NtermProteinFull
core.io.pdb.file_data: [ WARNING ] discarding 6 atoms at position 241 in file potra_pep_dock.pdb. Best match rsd_type: LEU_p:CtermProteinFull
core.conformation.Conformation: [ WARNING ] missing heavyatom: OXT on residue SER_p:CtermProteinFull 231
core.conformation.Conformation: [ WARNING ] missing heavyatom: OXT on residue LEU_p:CtermProteinFull 241
core.io.pdb.file_data: [ WARNING ] can't find atom for res 1 atom CP (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 1 atom CQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 1 atom OCP (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 1 atom 1HQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 1 atom 2HQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 1 atom 3HQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 231 atom CS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 231 atom NR (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 231 atom 1HS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 231 atom 2HS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 231 atom 3HS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 232 atom CP (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 232 atom CQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 232 atom OCP (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 232 atom 1HQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 232 atom 2HQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 232 atom 3HQ (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 241 atom CS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 241 atom NR (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 241 atom 1HS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 241 atom 2HS (trying to set temp)
core.io.pdb.file_data: [ WARNING ] can't find atom for res 241 atom 3HS (trying to set temp)


N-terminal residue with ACE cap

ATOM 1 N THR A 1 -31.488 3.511 18.328 1.00 78.37 N
ATOM 2 CA THR A 1 -30.945 2.436 19.202 1.00 76.50 C
ATOM 3 C THR A 1 -31.173 1.033 18.616 1.00 73.96 C
ATOM 4 O THR A 1 -32.122 0.812 17.851 1.00 74.65 O
ATOM 5 CP THR A 1 -31.290 4.730 18.837 1.00 78.37 C
ATOM 6 CQ THR A 1 -31.845 5.829 17.943 1.00 78.37 C
ATOM 7 OCP THR A 1 -30.777 4.920 19.806 1.00 78.37 O
ATOM 8 H THR A 1 -31.949 3.341 17.458 1.00 78.37 H
ATOM 9 HA THR A 1 -29.964 2.629 19.219 1.00 76.50 H
ATOM 10 1HQ THR A 1 -31.686 6.731 18.344 1.00 78.37 H
ATOM 11 2HQ THR A 1 -32.831 5.721 17.812 1.00 78.37 H
ATOM 12 3HQ THR A 1 -31.412 5.812 17.042 1.00 78.37 H
ATOM 13 CB THR A 1 -31.560 2.491 20.638 1.00 77.22 C
ATOM 14 HB THR A 1 -31.611 3.438 20.955 1.00 77.22 H
ATOM 15 OG1 THR A 1 -30.672 1.831 21.554 1.00 77.47 O
ATOM 16 HG1 THR A 1 -31.122 1.684 22.386 1.00 77.47 H
ATOM 17 CG2 THR A 1 -32.989 1.839 20.701 1.00 76.74 C
ATOM 18 1HG2 THR A 1 -33.363 1.885 21.627 1.00 76.74 H
ATOM 19 2HG2 THR A 1 -32.956 0.877 20.430 1.00 76.74 H
ATOM 20 3HG2 THR A 1 -33.625 2.309 20.089 1.00 76.74 H


C-terminal residue with NME cap

ATOM 3918 N SER A 231 22.277 37.377 -10.697 1.00 70.49 N
ATOM 3919 CA SER A 231 22.354 38.825 -10.930 1.00 73.91 C
ATOM 3920 C SER A 231 23.797 39.317 -11.192 1.00 75.99 C
ATOM 3921 O SER A 231 24.785 38.668 -10.791 1.00 76.11 O
ATOM 3922 NR SER A 231 24.016 40.378 -11.812 1.00 75.99 N
ATOM 3923 CS SER A 231 25.368 40.839 -12.057 1.00 75.99 C
ATOM 3924 H SER A 231 22.041 37.038 -9.786 1.00 70.49 H
ATOM 3925 HA SER A 231 21.809 38.952 -11.759 1.00 73.91 H
ATOM 3926 HR SER A 231 23.245 40.920 -12.145 1.00 75.99 H
ATOM 3927 1HS SER A 231 25.366 41.701 -12.564 1.00 75.99 H
ATOM 3928 2HS SER A 231 25.883 40.169 -12.591 1.00 75.99 H
ATOM 3929 3HS SER A 231 25.857 40.989 -11.198 1.00 75.99 H
ATOM 3930 CB SER A 231 21.748 39.604 -9.746 1.00 75.08 C
ATOM 3931 1HB SER A 231 21.903 40.586 -9.849 1.00 75.08 H
ATOM 3932 2HB SER A 231 20.767 39.428 -9.668 1.00 75.08 H
ATOM 3933 OG SER A 231 22.351 39.203 -8.525 1.00 77.89 O
ATOM 3934 HG SER A 231 21.678 39.113 -7.851 1.00 77.89 H


I have also built a peptide with the abinitio protocol and supplied Rosetta with a FASTA file, where I defined the terminal residues as N_acetylated and C_methylamidated, respectively. As expected, the abinitio protocol built a peptide with the requested terminal modifications.

I[ILE_p:N_acetylated]HWTMGPTQL[LEU_p:C_methylamidated]

This peptide was then used as input for FlexPepDocking, but the same warnings were printed as shown above. The names of the ACE/NME atoms are the same as in the two terminal residues listed above.

Here is the script I am using for the prepacking phase of FlexPepDocking:

#!csh
setenv rosetta3Dir /share/apps/rosetta3.5
setenv rosetta3DB ${rosetta3Dir}/rosetta_database
${rosetta3Dir}/rosetta_source/bin/FlexPepDocking.linuxgccdebug
-in:path:database ${rosetta3DB}
-in:file:s ${1}
-out:file:o ${2}
-constant_seed
-run:seed_offset ${3}
-flexpep_prepack
-ex1
-ex2aro
-receptor_chain A
-peptide_chain B


I hope you can tell me what’s wrong, and what I can do to enable Rosetta to correctly recognize the terminal modifications.

Thanks a lot for your help!

P.S.: I have also noticed a discrepancy between the centroid and fa_standard residue patches N_acetylated and C_methylamidated. I don’t know why “NOT VARIANT_TYPE UPPER_TERMINUS” is commented out in the centroid patch, but active in the fa_standard patch variant. Maybe this is somehow related to my problems with the terminal modifications?

fa_standard

N_acetylated

BEGIN_SELECTOR
PROPERTY PROTEIN
NOT VARIANT_TYPE N_ACETYLATION
NOT VARIANT_TYPE UPPER_TERMINUS
NOT VARIANT_TYPE LOWER_TERMINUS
NOT VARIANT_TYPE CUTPOINT_UPPER
NOT VARIANT_TYPE CUTPOINT_LOWER
NOT VARIANT_TYPE ACETYLATED_NTERMINUS
END_SELECTOR


C_methylamidated

BEGIN_SELECTOR
PROPERTY PROTEIN
NOT VARIANT_TYPE C_METHYLAMIDATION
NOT VARIANT_TYPE UPPER_TERMINUS
#NOT VARIANT_TYPE LOWER_TERMINUS
NOT VARIANT_TYPE CUTPOINT_UPPER
NOT VARIANT_TYPE CUTPOINT_LOWER
NOT PROPERTY PHOSPHONATE
END_SELECTOR


centroid


N_acetylated

BEGIN_SELECTOR
PROPERTY PROTEIN
NOT VARIANT_TYPE N_ACETYLATION
#NOT VARIANT_TYPE UPPER_TERMINUS
NOT VARIANT_TYPE LOWER_TERMINUS
NOT VARIANT_TYPE CUTPOINT_UPPER
NOT VARIANT_TYPE CUTPOINT_LOWER
END_SELECTOR


C_methylamidated

BEGIN_SELECTOR
PROPERTY PROTEIN
NOT VARIANT_TYPE C_METHYLAMIDATION
NOT VARIANT_TYPE UPPER_TERMINUS
#NOT VARIANT_TYPE LOWER_TERMINUS
NOT VARIANT_TYPE CUTPOINT_UPPER
NOT VARIANT_TYPE CUTPOINT_LOWER
END_SELECTOR


[Anonymous]

I’d recommend taking a look at the rosetta_database/chemical/residue_type_sets/fa_standard/patches.txt file. Check to make sure that the lines for the two patch files you’re using are not commented out. (That is, they don’t have a “#” character before them.)

Aside from that, it looks like you’re using the correct atom names for the modifications – although the forum eats spacing, so I’d recommend checking that the alignment of the atom names is correct.

[Anonymous]

Sorry, I should have included this information, too. In Rosetta 3.5 the two patch files are active by default in fa_standard/patches.txt as well as centroid/patches.txt. I have also checked whether the patches are read by Rosetta by including the two patch files with the “-extra_patch_fa” flag; this gives the following (expected) error message:

protocols.jd2.PDBJobInputter: filling pose from PDB test.pdb
ERROR: Attempting to add a residue type with name 'ALA_p:N_acetylated' but this name is already taken.

Thus, I assume that the patches are active.

@spacing:
I am very sure that the spacing of the atom names is correct. It’s the same as in the PDB file of the abinitio model of the peptide.

Any other idea what could be wrong?

[Anonymous]

Hi Omirus,
Just want to make it clear, only the peptide has NME at C-terminus and the receptor has ACE at N-terminus,only one modified residue for receptor and one for peptide, right?

[Anonymous]

No, in this case both, the receptor as well as the peptide, have ACE and NME caps. But for other peptides I want to add just ACE or NME; and other receptors might not need terminal modifications at all. I wrote a script to handle the construction of receptor-peptide complexes including terminal modifications as required.

[Anonymous]

I’m not sure why Rosetta isn’t recognizing the modification. Could you post an example input file as an attachment so I could play around with it? (You’ll have to change the extension to .txt in order to get around forum software restrictions.)

[Anonymous]

Hi Omirus,
I saw the pdb in Pymol and the peptide its broken. Connectivity doesn’t seems OK.

[Anonymous]

I am sorry. I have corrected my procedure for building the receptor-peptide complex. I have attached the complex again, but this time I have performed an energy minimization of the peptide to fix the geometry.

[Anonymous]

I have attached a PDB file with a receptor capped on both termini and a peptide capped on the N-terminus only. This complex (with the peptide placed randomly in close proximity to the receptor) is supposed to be a test case for flexible peptide docking.
Thanks a lot for your help!

please, find the corrected PDB file in my reply below

[Anonymous]

Hi All,
In my case only the peptide is Nterm-acetylated and Cterm-amidated. The C terminal modification is not recognized by Rosetta. I’m using 3.4 release version. For N_acetylated patch I added TYPES N_ACETYLATION LOWER_TERMINUS and it helps to recognize Nterm modification. For Cterm_amidated patch I added TYPES CTERM_AMIDATION UPPER TERMINUS , but it didn’t help.

Here is the Cterm ALA residue,
I deleted OXT atom and added NT,1HE2 and 2HE2 atoms.

ATOM 5694 N ALA B 369 7.565 -17.955 21.284 1.00 0.00 N
ATOM 5695 CA ALA B 369 8.334 -16.865 21.877 1.00 0.00 C
ATOM 5696 C ALA B 369 8.310 -16.945 23.384 1.00 0.00 C
ATOM 5697 O ALA B 369 7.273 -17.164 24.012 1.00 0.00 O
ATOM 5698 CB ALA B 369 7.804 -15.517 21.410 1.00 0.00 C
ATOM 5699 H ALA B 369 6.905 -18.461 21.858 1.00 0.00 H
ATOM 5700 HA ALA B 369 9.362 -16.815 21.519 1.00 0.00 H
ATOM 5701 1HB ALA B 369 8.386 -14.719 21.871 1.00 0.00 H
ATOM 5702 2HB ALA B 369 7.889 -15.448 20.326 1.00 0.00 H
ATOM 5703 3HB ALA B 369 6.759 -15.417 21.700 1.00 0.00 H
ATOM 5704 NT ALA B 369 9.564 -16.756 24.127 1.00 0.00 N
ATOM 5705 1HE2 ALA B 369 10.399 -16.576 23.562 1.00 0.00 H
ATOM 5706 2HE2 ALA B 369 9.702 -16.781 25.136 1.00 0.00 H

Thanks.

[Anonymous]

I didn’t use those flags as I got error saying “ERROR: Option matching -in:Ctermini not found in command line top-level context”.

[Ora Furman]

I have a similar case in Nterm-acetylated and Cterm-amidated. The modification is not recognized by Rosetta 3.5.
I modified the N terminal residue like this
ATOM 1 CP ILE A 1 8.090 13.768 -3.372 1.00 0.00 C
ATOM 2 OCP ILE A 1 7.951 12.760 -4.064 1.00 0.00 O
ATOM 3 CQ ILE A 1 9.375 14.583 -3.431 1.00 0.00 C
ATOM 4 1HQ ILE A 1 9.355 15.213 -4.321 1.00 0.00 H
ATOM 5 2HQ ILE A 1 10.225 13.904 -3.486 1.00 0.00 H
ATOM 6 3HQ ILE A 1 9.517 15.429 -2.252 1.00 0.00 H
ATOM 7 N ILE A 2 7.153 14.209 -2.539 1.00 0.00 N
ATOM 8 CA ILE A 2 5.869 13.531 -2.402 1.00 0.00 C
ATOM 9 C ILE A 2 4.762 14.301 -3.111 1.00 0.00 C
ATOM 10 O ILE A 2 4.537 15.479 -2.835 1.00 0.00 O

and the c-term looks like this
ATOM 132 2HD1 ILE A 8 -11.476 18.556 -2.601 1.00 0.00 H
ATOM 133 3HD1 ILE A 8 -12.666 17.988 -1.406 1.00 0.00 H
ATOM 135 N NT A 9 -15.576 15.081 -5.669 1.00 0.00 N
ATOM 136 H 1HE2 A 9 -16.788 15.240 -6.464 1.00 0.00 H
ATOM 137 H 2HE2 A 9 -15.520 14.326 -5.001 1.00 0.00 H

in the flag file, I put in
-in:Ntermini A
-in:Ctermini A

but after relax, it doesn’t give any error message, but generate a structure without the terminal modification. Anything I’m missing here? Should I explicitly add a patch file?

[Anonymous]

Hi smlewis,
I used 3.5 release, but the C terminal modification is not recognized. use of the flag -in:Ctermini B make the terminal residue recognized as ALA only, not the amidated alanine.

[Anonymous]

Hi Rocco,
alignment of the N of NT atom is correct.
About the NT atom Rosetta says,

core.io.pdb.file_data: (0) [ WARNING ] discarding 3 atoms at position 369 in file template_sele.pdb. Best match rsd_type: ALA
core.conformation.Conformation: (0) Failed to find a residue connection for residue 369 with connection point 2
core.io.pdb.file_data: (0) [ WARNING ] can’t find atom for res 369 atom NT (trying to set temp)
core.io.pdb.file_data: (0) [ WARNING ] can’t find atom for res 369 atom 1HE2 (trying to set temp)
core.io.pdb.file_data: (0) [ WARNING ] can’t find atom for res 369 atom 2HE2 (trying to set temp)

Thanks

[Ora Furman]

Hi rmoretti,thank you for catching the error in the format, the last residue looks like this

ATOM 115 N ILE A 8 -12.123 15.482 -5.642 1.00 0.00 N
ATOM 116 CA ILE A 8 -13.355 15.649 -4.881 1.00 0.00 C
ATOM 117 C ILE A 8 -14.542 15.905 -5.801 1.00 0.00 C
ATOM 118 O ILE A 8 -14.521 16.831 -6.613 1.00 0.00 O
ATOM 119 CB ILE A 8 -13.243 16.805 -3.869 1.00 0.00 C
ATOM 120 CG1 ILE A 8 -12.114 16.533 -2.872 1.00 0.00 C
ATOM 121 CG2 ILE A 8 -14.563 17.005 -3.142 1.00 0.00 C
ATOM 122 CD1 ILE A 8 -11.785 17.711 -1.985 1.00 0.00 C
ATOM 123 H ILE A 8 -11.819 16.226 -6.255 1.00 0.00 H
ATOM 124 HA ILE A 8 -13.608 14.729 -4.354 1.00 0.00 H
ATOM 125 HB ILE A 8 -12.978 17.718 -4.402 1.00 0.00 H
ATOM 126 1HG1 ILE A 8 -12.422 15.689 -2.256 1.00 0.00 H
ATOM 127 2HG1 ILE A 8 -11.231 16.256 -3.449 1.00 0.00 H
ATOM 128 1HG2 ILE A 8 -14.466 17.825 -2.431 1.00 0.00 H
ATOM 129 2HG2 ILE A 8 -15.343 17.242 -3.864 1.00 0.00 H
ATOM 130 3HG2 ILE A 8 -14.828 16.092 -2.609 1.00 0.00 H
ATOM 131 1HD1 ILE A 8 -10.976 17.442 -1.305 1.00 0.00 H
ATOM 132 2HD1 ILE A 8 -11.476 18.556 -2.601 1.00 0.00 H
ATOM 133 3HD1 ILE A 8 -12.666 17.988 -1.406 1.00 0.00 H
ATOM 135 NT ILE A 8 -15.576 15.081 -5.669 1.00 0.00 N
ATOM 136 1HE2 ILE A 8 -16.788 15.240 -6.464 1.00 0.00 H
ATOM 137 2HE2 ILE A 8 -15.520 14.326 -5.001 1.00 0.00 H

in the flag file, I put
-residues:patch_selectors CTERM_AMIDATION
-in:Ctermini A
but still rosetta doesn’t recogniz the last three atoms, NT and 1HE2 and 2HE2. Anything else I’m missing? Thanks very much.

[Anonymous]

You have to stick to Rosetta’s atom naming conventions. These two terminal modification patches are automatically applied by Rosetta, if the input structure already contains these modifications and they match the required atom names. You do not have to select them via the “patch_selectors” option.
If you want to know the required atom names of other modifications, just perform a quick abinitio modelling (of an arbitrary peptide/small protein), where you select the residue patches in the FASTA file (example given below); then analyze the atom names in the PDB output and modify the terminal residues in the PDB file of your protein of interest accordingly.

>abinitio test peptide
M[MET_p:N_acetylated]WDPLNQTLIHCNS[SER_p:C_methylamidated]


Here is an example of the format of the N-terminal acetylation:

ATOM 4000 N THR B 258 16.064 -2.321 32.177 1.00 0.00
ATOM 4001 CA THR B 258 17.306 -1.770 31.649 1.00 0.00
ATOM 4002 C THR B 258 18.472 -2.729 31.865 1.00 0.00
ATOM 4003 O THR B 258 19.591 -2.472 31.421 1.00 0.00
ATOM 4004 CP THR B 258 15.043 -1.482 31.988 1.00 0.00
ATOM 4005 CQ THR B 258 13.746 -2.057 32.539 1.00 0.00
ATOM 4006 OCP THR B 258 15.131 -0.475 31.484 1.00 0.00
ATOM 4007 CB THR B 258 17.642 -0.414 32.297 1.00 0.00
ATOM 4008 OG1 THR B 258 18.773 0.167 31.636 1.00 0.00
ATOM 4009 CG2 THR B 258 17.961 -0.594 33.774 1.00 0.00
ATOM 4010 H THR B 258 15.986 -3.213 32.623 1.00 0.00
ATOM 4011 HA THR B 258 17.150 -1.659 30.667 1.00 0.00
ATOM 4012 1HQ THR B 258 12.984 -1.425 32.396 1.00 0.00
ATOM 4013 2HQ THR B 258 13.820 -2.231 33.521 1.00 0.00
ATOM 4014 3HQ THR B 258 13.518 -2.921 32.091 1.00 0.00
ATOM 4015 HB THR B 258 16.787 0.252 32.189 1.00 0.00
ATOM 4016 HG1 THR B 258 18.567 0.300 30.707 1.00 0.00
ATOM 4017 1HG2 THR B 258 18.195 0.375 34.215 1.00 0.00
ATOM 4018 2HG2 THR B 258 17.098 -1.025 34.282 1.00 0.00
ATOM 4019 3HG2 THR B 258 18.816 -1.259 33.883 1.00 0.00

Here is an example of the format of the C-terminal methylamidation:

ATOM 4220 N GLN B 272 11.213 2.094 34.116 1.00 0.00
ATOM 4221 CA GLN B 272 10.940 2.132 35.547 1.00 0.00
ATOM 4222 C GLN B 272 9.461 1.893 35.833 1.00 0.00
ATOM 4223 O GLN B 272 8.629 1.938 34.927 1.00 0.00
ATOM 4224 NR GLN B 272 9.141 1.641 37.098 1.00 0.00
ATOM 4225 CS GLN B 272 7.736 1.414 37.370 1.00 0.00
ATOM 4226 CB GLN B 272 11.371 3.477 36.138 1.00 0.00
ATOM 4227 CG GLN B 272 12.866 3.737 36.070 1.00 0.00
ATOM 4228 CD GLN B 272 13.244 5.094 36.632 1.00 0.00
ATOM 4229 OE1 GLN B 272 12.377 5.896 36.992 1.00 0.00
ATOM 4230 NE2 GLN B 272 14.542 5.362 36.705 1.00 0.00
ATOM 4231 H GLN B 272 10.620 2.603 33.493 1.00 0.00
ATOM 4232 HA GLN B 272 11.478 1.387 35.943 1.00 0.00
ATOM 4233 HR GLN B 272 9.830 1.610 37.822 1.00 0.00
ATOM 4234 1HS GLN B 272 7.583 1.232 38.342 1.00 0.00
ATOM 4235 2HS GLN B 272 7.393 0.629 36.853 1.00 0.00
ATOM 4236 3HS GLN B 272 7.188 2.212 37.117 1.00 0.00
ATOM 4237 1HB GLN B 272 10.836 4.250 35.586 1.00 0.00
ATOM 4238 2HB GLN B 272 11.044 3.483 37.178 1.00 0.00
ATOM 4239 1HG GLN B 272 13.606 3.015 36.413 1.00 0.00
ATOM 4240 2HG GLN B 272 12.894 3.794 34.981 1.00 0.00
ATOM 4241 1HE2 GLN B 272 14.852 6.243 37.067 1.00 0.00
ATOM 4242 2HE2 GLN B 272 15.212 4.685 36.400 1.00 0.00


[Anonymous]

Hi Omirus,
For abinitio of the peptide M[MET_p:N_acetylated]WDPLNQTLIHCNS[SER_p:Cterm_amidated] I got error saying ERROR: unrecognized residue name ‘CYS_p:Cterm_amidated’ . Am I missing something here?

[Anonymous]

Looking at the code, I’m not quite sure what is supposed to be going on here. The main issue is that the PDB loading code is seeing that the residues are supposed to be chain termini, and is discarding the acetylated and methamidated patched residues because they’re not listed as proper chain termini.

I think if you edit the TYPES lines (these are different from the NOT VARIANT lines) of the respective patches to something like the following (i.e. adding the appropriate variant designation to those types), things should load appropriately.

TYPES C_METHYLAMIDATION UPPER_TERMINUS

and

TYPES N_ACETYLATION LOWER_TERMINUS

I’m not sure if that’s the best way of doing it, as when I tried it I got a memory error because of it. That was only after the simple test protocol had completed and gave me output, though, so the memory error might not be severe. I’ll talk to others and see what the problem is.

[Anonymous]

Thanks a lot for taking a deeper look at this issue!

I tried your suggested modifications and also got a memory error, when running the prepacking phase of FlexPepDock (release build); the debug build printed the following before aborting:

core.chemical.ResidueTypeSet: Finished initializing fa_standard residue type set. Created 6242 residue types
FlexPepDocking.linuxgccdebug: src/utility/vectorL.hh:362: typename std::vector::reference utility::vectorL<, T, A>::operator[](typename utility::vectorL_IndexSelector<(L >= 0)>::index_type) [with long int L = 1l, T = core::chemical::ResConnID, A = std::allocator]: Assertion `vectorL_ZeroSelector< L != 0 >::ge( i, l_ )' failed.
Got some signal... It is:6
Process was aborted!


[Anonymous]

Turns out there’s flags that will turn off the termini assumption. -in:Ntermini and -in:Ctermini take a string of chain designations for which Rosetta will try different chain terminations. For example, you would add the flags “-in:Ntermini AB -in:Ctermini A” to your commandline to tell Rosetta you have modifications on the N-termini of chains A and B, and the C termini of chain A. You do *not* need to modify your database files to use these flags.

Turns out the memory error is related to something else. Rosetta apparently doesn’t like a chain with an N-terminal modification directly following a chain with a C-terminal modification. If you can flip your chains in your PDB, such that your B chain comes before your A chain, that should fix things (as your B chain doesn’t have a C-terminal modification.) This might mess up your foldtree and the assumptions of your downstream protocol, though. (Because although the PDB chain IDs can stay the same, the chain numbering and jump relationships will change.) If flipping the chains doesn’t work, I’d suggest removing (or just not loading) the C-terminal modification for chain A. If it’s far enough (>6 Ang) from the binding site, the presence/absence of the modification shouldn’t make all that much of a difference to the results.

[Anonymous]

Yes, it’s working! Thank you very much! It’s just too bad that currently Rosetta can not deal with all combinations of terminal modifications. I hope in future releases this bug will be fixed. In the meantime I will try to work around it.

FYI, when I try to extract the models from the binary silent file, I get the following message from ‘extract_pdbs’:

core.io.silent: Finished reading 2 structures from default.out
core.pose: Residue following X, Z, or an upper terminus is _not_ a lower terminus type! Continuing ...
core.pose: Residue following X, Z, or an upper terminus is _not_ a lower terminus type! Continuing ...

The PDBs are still extracted, but to my surprise each residue is followed by a TER and has its own chain ID. Anyway, I can easily fix this with a script. I just wanted to inform you about this little bug.

[Anonymous]

Yeah, there seems to be a bug with putting together the foldtree in these sorts of situations. It’s been fixed in the development version, but that was after the 3.5 release.

[Anonymous]

To clarify…is this also using the -Ntermini / -Ctermini flags Rocco mentions here: https://www.rosettacommons.org/node/3331#comment-6385 (a few posts up)?

[Anonymous]

Rereading Rocco’s posts (and checking the code), that flag isn’t IN the 3.4 release. Can you try updating to 3.5 (or better yet, the most recently weekly)?

[Anonymous]

You’ve have several problems with your input PDB file. The first is that you have extra N & H symbols after the atom sequence number, and you’ve omitted the residue type name. The second is that you’ve made the amidation a separate residue. Rosetta will be expecting the c-terminal modification to be part of the c-terminal residue, not a separate residue.

[Anonymous]

What, if anything, is Rosetta saying about that NT atom? It should be commenting that it’s discarding those heavy atom coordinates (try adding “-out:level 400” to the commandline to increase the tracer amounts.)

Just to double check, what’s your atom name alignment? The “N” of the NT should fall in the same vertical column as the N/O/C of the other atom names. If it’s misaligned, Rosetta won’t recognize it.

[Anonymous]

The name of this specific C-terminal residue patch is “Cterm_amidation” – at least in Rosetta 3.5. Look it up in “rosetta_database/chemical/residue_type_sets/fa_standard/patches/Cterm_amidation.txt”.

[Author]

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