My aim is to model single chain protein from a 2 chain template with 2 ligands ( PLP).following the advance tutorial I have edited template PDB ( i.e.1IAX) as per my requirement.I have deleted chain B in the ligand which will interact with it.now template is look like this-

ATOM 2020 N SER A 277 6.836 50.703 64.530 1.00 50.02 N ATOM 2021 CA SER A 277 5.736 50.683 65.488 1.00 52.57 C ATOM 2022 C SER A 277 6.158 50.953 66.920 1.00 53.76 C ATOM 2023 O SER A 277 5.656 51.877 67.558 1.00 53.78 O ATOM 2024 CB SER A 277 5.023 49.340 65.432 1.00 53.52 C ATOM 2025 OG SER A 277 4.399 49.181 64.168 1.00 60.54 O ATOM 2026 N LYS A 278 7.083 50.145 67.425 1.00 55.18 N ATOM 2027 CA LYS A 278 7.540 50.304 68.795 1.00 56.49 C ATOM 2028 C LYS A 278 8.430 51.529 69.009 1.00 56.08 C ATOM 2029 O LYS A 278 8.170 52.321 69.915 1.00 56.54 O ATOM 2030 CB LYS A 278 8.218 49.013 69.274 1.00 59.46 C ATOM 2031 CG LYS A 278 7.268 47.807 69.271 1.00 63.38 C ATOM 2032 CD LYS A 278 6.048 48.015 70.191 1.00 69.44 C ATOM 2033 CE LYS A 278 4.984 46.952 69.962 1.00 73.69 C ATOM 2034 NZ LYS A 278 5.598 45.765 69.377 1.00 82.22 N TER 2035 LYS A 278 HETATM 6623 S SO4 600 2.001 43.793 71.949 0.40 47.42 S HETATM 6624 O1 SO4 600 1.250 44.661 72.856 1.00 49.68 O HETATM 6625 O2 SO4 600 3.441 43.960 72.132 1.00 48.63 O HETATM 6626 O3 SO4 600 1.702 42.399 72.263 1.00 51.95 O HETATM 6627 O4 SO4 600 1.623 44.087 70.571 1.00 47.72 O HETATM 6633 N1 PLP A 500 6.942 41.456 67.024 1.00 91.96 N HETATM 6634 C2 PLP A 500 7.237 41.844 68.301 1.00 92.40 C HETATM 6635 C2A PLP A 500 8.277 41.064 69.104 1.00 91.84 C HETATM 6636 C3 PLP A 500 6.579 42.967 68.880 1.00 91.52 C HETATM 6637 O3 PLP A 500 6.876 43.351 70.176 1.00 90.45 O HETATM 6638 C4 PLP A 500 5.613 43.677 68.118 1.00 90.22 C HETATM 6639 C4A PLP A 500 4.901 44.884 68.713 1.00 86.77 C HETATM 6640 C5 PLP A 500 5.327 43.248 66.794 1.00 91.55 C HETATM 6641 C6 PLP A 500 6.014 42.124 66.269 1.00 92.40 C HETATM 6642 C5A PLP A 500 4.287 43.977 65.936 1.00 92.05 C HETATM 6643 O4P PLP A 500 4.919 44.983 65.137 1.00 91.48 O HETATM 6644 P PLP A 500 3.850 45.746 64.212 0.40 89.98 P HETATM 6645 O1P PLP A 500 2.942 46.559 65.059 1.00 90.28 O HETATM 6646 O2P PLP A 500 3.052 44.763 63.428 1.00 89.69 O HETATM 6647 O3P PLP A 500 4.581 46.636 63.284 1.00 90.10 O HETATM 6663 O HOH 1 -2.199 65.598 27.605 1.00 37.63 O HETATM 6664 O HOH 2 13.146 43.997 69.980 1.00 28.90 O HETATM 6665 O HOH 3 3.514 34.147 84.461 1.00 40.65 O HETATM 6666 O HOH 4 14.245 36.668 40.667 1.00 33.18 O HETATM 6667 O HOH 5 6.748 58.168 71.500 1.00 37.09 O HETATM 6668 O HOH 6 -3.541 20.765 51.638 1.00 34.79 O HETATM 6669 O HOH 7 -4.102 61.235 21.691 1.00 38.94 O HETATM 6670 O HOH 8 10.326 70.845 65.572 1.00 35.43 O HETATM 6671 O HOH 9 -4.995 51.193 45.379 1.00 38.86 O HETATM 6672 O HOH 10 -6.557 33.928 65.811 1.00 39.72 O HETATM 6673 O HOH 11 -1.108 49.863 77.220 1.00 41.22 O HETATM 6674 O HOH 12 -26.403 30.845 53.499 1.00 43.93 O HETATM 6675 O HOH 13 -6.662 49.811 60.632 1.00 20.93 O HETATM 6676 O HOH 14 0.524 46.344 51.157 1.00 30.77 O HETATM 6677 O HOH 15 4.444 27.533 53.723 1.00 45.47 O HETATM 6678 O HOH 16 -9.377 50.142 40.921 1.00 38.67 O HETATM 6679 O HOH 17 16.732 42.270 53.224 1.00 43.17 O HETATM 6680 O HOH 18 36.203 41.041 67.666 1.00 41.06 O HETATM 6681 O HOH 19 -23.753 42.588 51.308 1.00 39.07 O CONECT 2034 6639 CONECT 5345 6654 CONECT 6623 6624 6625 6626 6627 CONECT 6624 6623 CONECT 6625 6623 CONECT 6626 6623 CONECT 6627 6623 CONECT 6628 6629 6630 6631 6632 CONECT 6629 6628 CONECT 6630 6628 CONECT 6631 6628 CONECT 6632 6628 CONECT 6633 6634 6641 CONECT 6634 6633 6635 6636 CONECT 6635 6634 CONECT 6636 6634 6637 6638 CONECT 6637 6636 CONECT 6638 6636 6639 6640 CONECT 6639 2034 6638 CONECT 6640 6638 6641 6642 CONECT 6641 6633 6640 CONECT 6642 6640 6643 CONECT 6643 6642 6644 CONECT 6644 6643 6645 6646 6647 CONECT 6645 6644 CONECT 6646 6644 CONECT 6647 6644 CONECT 6648 6649 6656 CONECT 6649 6648 6650 6651 CONECT 6650 6649 CONECT 6651 6649 6652 6653 CONECT 6652 6651 CONECT 6653 6651 6654 6655 CONECT 6654 5345 6653 CONECT 6655 6653 6656 6657 CONECT 6656 6648 6655 CONECT 6657 6655 6658 CONECT 6658 6657 6659 CONECT 6659 6658 6660 6661 6662 CONECT 6660 6659 CONECT 6661 6659 CONECT 6662 6659 MASTER 342 0 4 47 25 0 0 6 6679 2 42 66 END II have made alignment in PIR using chain breaks "/" and block residues "." for SO4,PLP AND HOH.My alignment looks like this:

>P1;TvLDH sequence:TvLDH: : : : ::: 0.00: 0.00 MRIYGEEHPNQQILSRIATNDGHGENSSYFDGWKAYEKDPFHLTDNPTGVIQMGLAENQL SLDLIRDWMKKNPQASICTEEGVSEFKAIANFQDYHGLPTFRKAIAQFMEKVRGGRTRFD PDRIVMSGGATGAQETIAFCLADPGEAFLIPTPYYPGFDR-FRWRTGVQLLPIHCHSSNK FKITQAALETAYRKARNSHIRVKGILVTNPSNPLGTTMDRETLRTLVSFVNEKRMHLVCD EIFSGTAFDKPSYVSVSEVIEDE--PYCDRDLIHIAYSLSKDLGVPGFRVGVIYSYNDAV VSCARKMSSFGLVSSQTQHLLASMLGDEELTTSFLATSRTGLCGRRRVFTDGLKRVGIHC LDGNAGLFCWMDLRPLLKEATVEAELRLWRVIINDVKLNISPGSSFHCSEPGWFRVCFAN MDDTAMKIALRRIESFVYRENDAAVQAKNKRRWDEALRLSLPRRRFEDPTIMTPHLMSPH SPLVQAAT/..* >P1;TvLDH_model structureX:TvLDH-loop:1 : :485 : ::: 0.00: 0.00 MRIYGEEHPNQQILSRIATNDGHGENSSYFDGWKAYEKDPFHLTDNPTGVIQMGLAENQL SLDLIRDWMKKNPQASICTEEGVSEFKAIANFQDYHGLPTFRKAIAQFMEKVRGGRTRFD PDRIVMSGGATGAQETIAFCLADPGEAFLIPTPYYPGFDR-FRWRTGVQLLPIHCHSSNK FKITQAALETAYRKARNSHIRVKGILVTNPSNPLGTTMDRETLRTLVSFVNEKRMHLVCD EIFSGTAFDKPSYVSVSEVIEDE--PYCDRDLIHIAYSLSKDLGVPGFRVGVIYSYNDAV VSCARKMSSFGLVSSQTQHLLASMLGDEELTTSFLATSRTGLCGRRRVFTDGLKRVGIHC LDGNAGLFCWMDLRPLLKEATVEAELRLWRVIINDVKLNISPGSSFHCSEPGWFRVCFAN MDDTAMKIALRRIESFVYRENDAAVQAKNKRRWDEALRLSLPRRRFEDPTIMTPHLMSPH SPLVQAAT/--* >P1;1IAX structureX:1IAX: 126 :A : 500 : :undefined:undefined:-1.00:-1.00 ------------------------------------------------------------ ------------------------------------------------------------ --------GATGANETIIFCLADPGDAFLVPSPYYPAFNRDLRWRTGVQLIPIHCESSNN FKITSKAVKEAYENAQKSNIKVKGLILTNPSNPLGTTLDKDTLKSVLSFTNQHNIHLVCD EIYAATVFDTPQFVSIAEILDEQEMTYCNKDLVHIVYSLSK------------------- ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ --------/..*

Then I used advanced example as a template to write my input script:

from modeller import * from modeller.automodel import *

class mymodel(automodel): def special_restraints(self, aln): rsr = self.restraints for ids in (('NZ:278:A','C4A:500:A')):

atoms = [self.atoms[i] for i in ids] rsr.add(forms.upper_bound(group=physical.upper_distance, feature=features.distance(*atoms), mean=3.5, stdev=0.1))

env = environ() env.io.hetatm = True a = mymodel(env, alnfile='align-ligand.ali', knowns=('TvLDH_model','1IAX'), sequence='TvLDH') a.starting_model = 1 a.ending_model = 5 a.make()

then program starts calculating, and then I always get :

check_ali___> Checking pairwise structural superpositions.

Equivalent CA pairs with distance difference larger than 6.0 angstroms:

ALN_POS TMPL1 TMPL2 RID1 RID2 NAM1 NAM2 DIST ---------------------------------------------------- END OF TABLE

check_ali___> Checking the sequence-structure alignment.

Implied target CA(i)-CA(i+1) distances longer than 8.0 angstroms:

ALN_POS TMPL RID1 RID2 NAM1 NAM2 DIST ---------------------------------------------- END OF TABLE read_to_681_> topology.submodel read from topology file: 3

getf_______W> RTF restraint not found in the atoms list: residue type, indices: 17 485 atom names : C +N atom indices : 3847 0

getf_______W> RTF restraint not found in the atoms list: residue type, indices: 17 485 atom names : C CA +N O atom indices : 3847 3843 0 3848

fndatmi_285W> Only 153 residues out of 155 contain atoms of type CA (This is usually caused by non-standard residues, such as ligands, or by PDB files with missing atoms.) mdtrsr__446W> A potential that relies on one protein is used, yet you have at least one known structure available. MDT, not library, potential is used. iup2crm_280W> No topology library in memory or assigning a BLK residue. Default CHARMM atom type assigned: S --> S This message is written only for the first such atom. 20 atoms in HETATM residues constrained to protein CA atoms within 10.00 angstroms 20 atoms in residues without defined topology constrained to be rigid bodies indxatm_278E> No ":" in ATOM:RESID[:CHAINID] atom identifier: N

but when I used model-ligand.py command it generate model including ligands successfully. So my questions are: 1. What am I doing wrong with model-multiple-hetero.py command? 2.how 2 solve this problem??

What do you think? Thank you for your suggestions.Sorry for my bad english. sanjay

sanjay Kumar singh kolkata Bose Institute Department of Botany Main Campus 93\1 A.P.C.Road Kolkata 700 009 India