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== Task description == The full description of this task can be found [[Task_homologyModelling|here]]. In this task we are going to learn more about several methods of homology modelling. There exists only a small number of known protein structures. Therefore if someone wants to predict the structure of a somehow new protein (newly sequenced, a mutant, etc.). He could use known structures to calculate a model of the unknown structure. Homology modelling bases on an alignment between the target-sequence and one or more template structures. The aligned coordinates are directly used for the model. That is why it is important to select a good template and alignment. == Calculation of models == === Overview of available homologous structures === ==== Search ==== We used hhsearch with the standard parameter to find homologous structures of our protein. The following command was executed: * ./hhsearch -i reference_pah_aa.fasta -d pdb70.db -b 500 -o hhsearch.out We received the following hits: {| border="1" |- ! No. ! PDB ID ! Description ! Prob ! E-Value ! P-Value ! Score ! SS ! Cols ! Query HMM ! Template HMM ! Residues ! Sequence Identity |- | 1 | 1phz_A | Protein (phenylalanine | 1 | 1 | 1 | 1084.4 | 0 | 429 | 1-429 | 1-429 | (429) | 92% |- | 2 | 1j8u_A | Phenylalanine-4-hydroxy | 1 | 1 | 1 | 894.5 | 0 | 325 | 103-427 | 1-325 | (325) | 100% |- | 3 | 1toh_A | Tyroh tyrosine hydroxy | 1 | 1 | 1 | 890.7 | 0 | 342 | 111-452 | 2-343 | (343) | 60% |- | 4 | 1mlw_A | Tryptophan 5-monooxygen | 1 | 1 | 1 | 804.2 | 0 | 300 | 116-415 | 2-301 | (301) | 66% |- | 5 | 1ltz_A | Phenylalanine-4-hydroxy | 1 | 1 | 1 | 504.9 | 0 | 265 | 144-414 | 2-269 | (297) | 30% |- | 6 | 2v27_A | Phenylalanine hydroxyla | 1 | 1 | 1 | 471.1 | 0 | 254 | 167-424 | 4-271 | (275) | 30% |- | 7 | 2qmx_A | Prephenate dehydratase; | 1 | 1 | 1 | 70.0 | 0 | 53 | 33-85 | 199-251 | (283) | 40% |- | 8 | 2qmw_A | PDT prephenate dehydra | 1 | 1 | 1 | 66.1 | 0 | 51 | 35-85 | 190-240 | (267) | 37% |- | 9 | 3luy_A | Probable chorismate mut | 1 | 1 | 1 | 66.0 | 0 | 53 | 33-85 | 207-259 | (329) | 28% |- | 10 | 1y7p_A | Hypothetical protein AF | 1 | 1 | 1 | 19.9 | 0 | 38 | 36-73 | 6-43 | (223) | 16% |} Important remark: the scores for Prob, E-Value and P-Value could not be calculated, that is why they are 1. ==== Template structure selection ==== We selected the following structures as our template structures: * > 60% sequence identity: 1phz * > 40% sequence identity: 1toh * < 40% sequence identity: 1ltz === Alignment Refinement === We used the reference for a search in PFAM. There were two PFAM-domains detected on the reference sequence: ACT and Biopterin_H Then we used the sequence of the three proteins 1PHZ, 1TOH and 1LTZ to run a search against PFAM and used the alignments with the HMMs of ACT and Biopterin_H and the alignment of the reference sequence with the HMMs of ACT and Biopterin_H as seeds for the improved alignments. The crucial parts of the reference sequence according to the annotation in UniProt was already aligned by the seeds. We predicted the secondary structure of the three sequences of the proteins (TODO ref... seems to be better to predict) and tried to extend the seeds. In PHZ a large gap could be filled by high sequence identity. Afterwards we deleted the unaligned ends of the reference sequence to improve the resulting model. The essential part of the protein, the domains, should now be better modeled. <code> Seeds by PFAM - ACT: {| border="1" ! REFERENCE | SLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKkDEYEFFTHLD-KRSL |- ! 1PHZ | SLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNkDEYEFFTYL------ |} Seeds by PFAM - Biopterin_H {| border="1" ! REFERENCE | PWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYK<br> THACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPE<br> PDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCL-S<br> EKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSE<br> IGILCSALQK |- ! 1PHZ | PWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYK<br> THACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPE<br> PDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCL-S<br> DKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT-------------<br> <nowiki>----------</nowiki> |- ! 1TOH | PWFPRKVSELDKC-----------DLDHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYA<br> THACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPE<br> PDCCHELLGHVPMLADRTFAQFSQDIGLASLGASD-EEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSL-S<br> EEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDE<br> LHTLAHALS- |- ! 1LTZ | ------------------------------------------------------PQPLDRYSAEDHATWATLYQRQCKLLP<br> GRACDEFLEGLERLEV----DADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQE<br> PDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKAlGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLdS<br> ASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQ----------------------------------------------<br> <nowiki>----------</nowiki> |} unadjusted alignment of 1PHZ {| border="1" ! REFERENCE | MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFF<br> THLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQ<br> FADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGF<br> RLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEK<br> LATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVR<br> NFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK |- ! PSI | CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEE<br> EECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHH<br> HHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCC<br> EEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHH<br> HHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHH<br> HHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC |- ! 1PHZ | ------------------GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFF<br> TYLDKRTKPVLGSIIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQ<br> FADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGF<br> RLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEK<br> LATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCLSDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVR<br> TFAATIPRPFSVRYDPYTQRVEVLDNT------------------------- |- ! PSI | ------------------CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEE<br> EEECCCCCCCHHHHHHHHHCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHH<br> HHHHCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCC<br> EEEECCCCCCHHHHHHHHHCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHH<br> HHHHEEEEEEEEEEEECCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHH<br> HHHHHCCCCCCCCCCCCCCEEEECCCC------------------------- |} unadjusted alignment of 1TOH {| border = "1" ! REFERENCE | MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFF<br> THLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQ<br> FADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGF<br> RLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEK<br> LATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVR<br> NFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK |- ! PSI | CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEE<br> EECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHH<br> HHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCC<br> EEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHH<br> HHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHH<br> HHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC |- ! 1PHZ | ------------------GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFF<br> TYLDKRTKPVLGSIIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQ<br> FADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGF<br> RLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEK<br> LATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCLSDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVR<br> TFAATIPRPFSVRYDPYTQRVEVLDNT------------------------- |- ! PSI | ------------------CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEE<br> EEECCCCCCCHHHHHHHHHCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHH<br> HHHHCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCC<br> EEEECCCCCCHHHHHHHHHCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHH<br> HHHHEEEEEEEEEEEECCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHH<br> HHHHHCCCCCCCCCCCCCCEEEECCCC------------------------- |} unadjusted alignment of 1LTZ {| border="1" ! REFERENCE | MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFF<br> THLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQ<br> FADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGF<br> RLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIE<br> KLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCL-SEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEK<br> VRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK |- ! PSI | CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEE<br> EECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHH<br> HHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCC<br> EEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCH HHHH<br> HHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHH CCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHH<br> HHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC |- ! 1LTZ | -----------------F--------------------------V-----V-----------------------------<br> <nowiki>--------PDITT-----RKNVG-----LSHDANDFTLP------QPLDR-------YSA--------------------</nowiki><br> <nowiki>---------------------EDHATWATLYQRQCKLLPGRACDEFLEGLERLE----VDADRVPDFNKLNEKLMAATGW</nowiki><br> KIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKALGALP<br> MLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLDSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQL---<br> <nowiki>---FDAD----FAPLY------LQLAD-AQPWG--AGDIAP------DDL--VL</nowiki> |- ! PSI | -----------------C--------------------------C-----C-----------------------------<br> <nowiki>--------CCCCC-----CCCCC-----CCCCCCCCCCC------CCCCC-------CCH--------------------</nowiki><br> <nowiki>---------------------HHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCC----CCCCCCCCCHHHHHHHHHHHCC</nowiki><br> EEEECCCCCCHHHHHHHHHCCCCCEEECCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCCCCHHH<br> HHHHHHEEEEEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCCCEEECCCHHHHHCCCCCCCCCCCCEEEECCHHHH---<br> <nowiki>---HHHC----CHHHH------HHHHH-CCCCC--CCCCCC------CCC--CC</nowiki> |} adjusted alignment of 1LTZ {| border="1" ! REFERENCE | PIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSR<br> DFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFG<br> LCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAAT |- ! PSI | CCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHH<br> HHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEE<br> EECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHH |- ! 1LTZ | PQPLDRYSAEDHATWATLYQRQCKLLPGRACDEFLEGLERLEV----DADRVPDFNKLNEKLMAATGWKIVAVPGLIPDD<br> VFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKAlGALPMLARLYWYTVEF<br> GLINTPAGMRIYGAGILSSKSESIYCLdSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQLFDADFAPL |- ! PSI | CCCCCCCCHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHHHCCEEEECCCCCCHHHHHH<br> HHHCCCCCEEECCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCCCCHHHHHHHHHEEEEEEEEEE<br> CCCCEEEECCCCCCCCCCCCCCCCCCCCEEECCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHCCHHHHHHH |} adjusted alignment of 1PHZ {| border="1" ! REFERENCE | GQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKIL<br> RHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVE<br> YMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGL<br> AFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGD<br> SIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYT<br> QRIEVLDNTQQ |- ! PSI | CCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHH<br> CCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCC<br> CCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHC<br> CCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCC<br> CEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCC<br> CEEEECCCHHH |- ! 1PHZ | GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNkDEYEFFTYLDKRTKPVLGSIIKSL<br> RNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVE<br> YTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGL<br> AFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKEG<br> DSIKAYGAGLLSSFGELQYCL-SDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDP<br> YTQRVEVLDNT |- ! PSI | CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEEEEECCCCCCCHHHHHHHH<br> HCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHHHHHHCCCCCCCCCCCCCC<br> CCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCCEEEECCCCCCHHHHHHHH<br> HCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCH-HHHHHHHHHEEEEEEEEEEEEC<br> CCCEEECCCCCCCCCCCCCCC-CCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHHHHHHHCCCCCCCCCCC<br> CCCEEEECCCC |} adjusted alignment of 1TOH {| border="1" ! REFERENCE | VPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSL<br> YKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMY<br> TPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYC<br> LSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSI<br> NSEIGILCSALQKIK |- ! PSI | CCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHH<br> HCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCC<br> CCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHH<br> HCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHH<br> HHHHHHHHHHHHHHC |- ! 1TOH | VPWFPRKVSELDKC-----------DLDHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGL<br> YATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMH<br> SPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASD-EEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLH<br> SL-SEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLE<br> GVQDELHTLAHALSA |- ! PSI | CCCCCCCCCCCCCC-----------CCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHHHHHHHH<br> HHHCCCHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHCCCEEEECCCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCC<br> CCCCCHHHHHHCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHCEEEEEEEEEEEEECCCCEECCCCCCCCCCHHCCC<br> CCCCCCCCCCCHHHHHCCCCCCCCCCCEEEEECCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCEECCCCHHHHHHHHHHH<br> HHHHHHHHHHHHHHC |} </code> === Homology modeling with Modeller === Modeller uses three files. The first one contains the used alignment in the PIR-format (see [http://salilab.org/modeller/manual/node463.html PIR FORMAT]). This file should at least contain the sequence of the target, the actual alignment can be calculated by Modeller or specified in this file. The second one is a python script, which tells Modeller which steps it has to perform. There are some examples in <code>/apps/modeller9.9/examples/automodel</code>. And the third file is the pdb-file of the used template. But python seems to be falsely configured on the virtual machines (at least the linux virtual machine with the non-sudo user). * The used fix of the python installation is described at the [[resource_software|software section]]. * The Modeller modules were still not importable by Python, that is why it was necessary to reinstall Modeller. The steps for this are described in [[resource_software|software section]]. In the automated workflow Modeller calculated the alignment by himself (see [http://salilab.org/modeller/tutorial/basic.html basic Modeller tutorial]). In the adjusted workfow we changed the alignmentfile to match our alignment. We prepared three files for the modeling with one template(e.g. for 1PHZ). * The alignment input file, which contains only the reference sequence: pah.ali. This file is the basis for Modellers own alignment to the template. <br> <code> >P1;PAH<br> sequence:reference::::::::<br>MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEEN<br>DVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKD<br>TVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPI<br>PRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQ<br>FLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGH<br>VPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLL<br>SSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATI<br>PRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK*</code> * We splitted the python script in order to be able to modify the alignment file, which was created by Modeller in the first part of the script and used by the second part of the script. * The python script, which tells Modeller to create an alignment <br><code>from modeller import *<br><br>env = environ()<br>aln = alignment(env)<br>mdl = model(env, file='../structures/1phz.pdb', model_segment=('FIRST:A','LAST:A'))<br>aln.append_model(mdl, align_codes='1phz', atom_files='../structures/1phz.pdb')<br>aln.append(file='pah.ali', align_codes='PAH')<br>aln.align2d()<br>aln.write(file='phz.ali', alignment_format='PIR')<br> </code> * The python script, which tells Modeller to create a model<br><code>from modeller.automodel import *<br><br>a = automodel(env, alnfile='phz.ali',<br> knowns='1phz', sequence='PAH',<br> assess_methods=(assess.DOPE, assess.GA341))<br>a.starting_model = 1<br>a.ending_model = 1<br>a.make()<br> </code> For the modeling with multiple templates we used the alignment file described above and a new script (see [http://salilab.org/modeller/tutorial/advanced.html advanced Modeller tutorial]). We used the sturctures of 1MLW, 1TOH, 1PHZ as templates * <code>from modeller import *<br>from modeller.automodel import *<br><br>env = environ()<br>aln = alignment(env)<br><br>path = '../structures/'<br>l = (path+'1MLW'+'.pdb', path+'1TOH'+'.pdb', path+'1PHZ'+'.pdb')<br><br>for (code) in l:<br> mdl = model(env, file=code, model_segment=('FIRST:A','LAST:A'))<br> aln.append_model(mdl, align_codes=code, atom_files=code)<br>aln.append(file='pah.ali', align_codes='PAH')<br>aln.align2d()<br>aln.write(file='mult.ali', alignment_format='PIR')<br><br>a = automodel(env, alnfile='mult.ali',<br> knowns=l, sequence='PAH',<br> assess_methods=(assess.DOPE, <br>ssess.GA341))<br>a.starting_model = 1<br>a.ending_model = 1<br>a.make()</code> === Homology modeling with Swissmodel === ==== Standard workflow ==== The standard workflow of Swissmodel is the automated mode. For this mode only the UniProt accession number or the amino acid sequence of the target protein is required. As an optional parameter it is possible to enter the template structure as well. However, if this field is left blank Swissmodel will search automatically for a suitable template. The input for all three models is as follows: {| border="1" |- ! Category ! Template ! Target ! Image |- | > 60% sequence identity | 1PHZ Chain: A | P00439 (Phenylalanine-4-hydroxylase) | [[File:1phz A template auto model.png|400px]] |- | > 40% sequence identity | 1TOH Chain: A | P00439 (Phenylalanine-4-hydroxylase) | [[File:1toh A template auto model.png|400px]] |- | < 40% sequence identity | 1LTZ Chain: A | P00439 (Phenylalanine-4-hydroxylase) | [[File:1ltz A template auto model.png|400px]] |} ==== Workflow with own alignment ==== The workflow of Swissmodel, where you can use your own alignment, is the automated mode. For this mode the alignment has to be specified. Swissmodel accepts the alignment in different formats. We have chosen FASTA. In a new window you have to specify which FASTA id is the target and which FASTA id is the template. For the template you have to specify the corresponding pdb-id with chain. Afterwards you have to check the updated alignment with respect to the pdb structure. The screenshots of the workflow for 1phz are shown below. {| border="1" |- ! 1. Specify the alignment ! 2. Specify target and template with structure ! 3. Checking of the adjusted alignment |- | [[File:Pah swiss 1.png|400px]] | [[File:Pah swiss 2.png|400px]] | [[File:Pah swiss 3.png|400px]] |} === Homology modelling with iTasser === [[Image:Pah itasser 1.png|thumb|top|Figure 1: screenshot of iTasser workflow with template restriction]] [[Image:Pah itasser 2.png|thumb|top|Figure 2: screenshot of iTasser workflow with alignment and template restriction]] [http://zhanglab.ccmb.med.umich.edu/I-TASSER/ ITasser] is a prediction server, which participated in several CASP competitions. It claims of itself to be the best one. The runtime of the jobs is approximately 24 to 48 hours. The server seems to receive a lot of jobs, that is why it is not allowed to add more than one job at a time from the same ip-address. Therefore it is probably not possible to run all six variants described in the task. iTasser offers several options. For the automated workflow we just specified a template to be used (a screenshot can be seen in figure 1). For the workflow with our adjusted alignment we specified the template with alignment (a screenshot can be seen in figure 2). The file which was needed for the workflow with the adjusted alignment contains the alignment in FASTA format and the ATOM coordinates of the template:<br> <code> * >TARGET<br>GQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTN<br>IIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNY<br>RHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLR<br>PVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYI<br>EKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFN<br>DAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQ<br>>1phy:A<br>GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFFTYLDKRTKPVLGS<br>IIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNY<br>RHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLR<br>PVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EY<br>IEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCL-SDKPKLLPLELEKTACQEYSVTEFQPLYYVAES<br>FSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT<br><br>ATOM 1 N GLY A 19 40.338 -7.649 17.712 1.00117.94 N <br>ATOM 2 CA GLY A 19 40.523 -8.622 16.594 1.00119.56 C <br>ATOM 3 C GLY A 19 39.585 -8.344 15.430 1.00119.47 C <br>ATOM 4 O GLY A 19 39.005 -9.256 14.849 1.00122.09 O <br>...<br>ATOM 3284 OG1 THR A 427 12.515 -6.231 40.981 1.00107.96 O <br> ATOM 3285 CG2 THR A 427 14.463 -5.654 39.679 1.00107.51 C <br> END </code> === Model-refinement with 3D-JigSaw === Without a reference structure it is hard to say which predicted model is the best. There are methods and servers, which try to rank several models. If their performance would be perfect, the modeling of proteins was a solved problem. But their performance is sometimes far from perfect. One of these severs is [http://bmm.cancerresearchuk.org/~populus/populus_submit.html 3D-Jigsaw]. In fact 3D-Jigsaw is a complete protein modeling server which takes the models and tries to improve them and rank them afterwards. The ranking of the models needs a lot of resources on the JigSaw server. Our goal is to select five of the six models for each group. Therefore we try to kick out models which are too similar. That is why we calculated the pairwise TMScore for the different models of one prediction class and selected only one member of a high-scoring pair. ====Selection of models==== =====1phz - sequence identity > 60 %===== {| border="1" ! ! modeller ! modeller adjusted ! swissmodel ! swissmodel adjusted ! iTasser ! iTasser adjusted |- ! modeller | 1.0000 | 0.0941 | 0.9773 | 0.0933 | 0.8911 | 0.0958 |- ! modeller adjusted | 0.0885 | 1.0000 | 0.0918 | 0.9808 | 0.0887 | 0.8141 |- ! swissmodel | 0.8845 | 0.0915 | 1.0000 | 0.0906 | 0.8883 | 0.0942 |- ! swissmodel adjusted | 0.0879 | 0.9808 | 0.0909 | 1.0000 | 0.0877 | 0.8181 |- ! iTasser | 0.8911 | 0.0937 | 0.9815 | 0.0932 | 1.0000 | 0.0959 |- ! iTasser adjusted | 0.0902 | 0.8141 | 0.0945 | 0.8181 | 0.0898 | 1.0000 |} The similarity between the models of modeller with adjusted alignment and swissmodel with adjusted alignment is too high. We decided to kick the model of modeller. ===== 1toh - sequence identity > 40 % ===== {| border="1" ! ! modeller ! modeller adjusted ! swissmodel ! swissmodel adjusted ! iTasser ! iTasser adjusted |- ! modeller | 1.0000 | 0.0941 | 0.7839 | 0.0929 | 0.5782 | 0.0910 |- ! modeller adjusted | 0.0759 | 1.0000 | 0.0928 | 0.9379 | 0.0812 | 0.7005 |- ! swissmodel | 0.5818 | 0.0926 | 1.0000 | 0.0893 | 0.6602 | 0.0878 |- ! swissmodel adjusted | 0.0750 | 0.9379 | 0.0895 | 1.0000 | 0.0792 | 0.7118 |- ! iTasser | 0.5782 | 0.1008 | 0.8902 | 0.0980 | 1.0000 | 0.0961 |- ! iTasser adjusted | 0.0733 | 0.7005 | 0.0881 | 0.7118 | 0.0779 | 1.0000 |} The similarity between the models of modeller with adjusted alignment and swissmodel with adjusted alignment is too high. We decided to kick the model of modeller. ===== 1ltz - sequence identity < 40 % ===== {| border="1" ! ! modeller ! modeller adjusted ! swissmodel ! swissmodel adjusted ! iTasser ! iTasser adjusted |- ! modeller | 1.0000 | 0.0898 | 0.8383 | 0.0889 | 0.4521 | |- ! modeller adjusted | 0.0589 | 1.0000 | 0.0661 | 0.9722 | 0.0592 | |- ! swissmodel | 0.4601 | 0.0695 | 1.0000 | 0.0689 | 0.4566 | |- ! swissmodel adjusted | 0.0577 | 0.9722 | 0.0656 | 1.0000 | 0.0601 | |- ! iTasser | 0.4521 | 0.0941 | 0.8266 | 0.0951 | 1.0000 | |- ! iTasser adjusted | | | | | | |} iTasser is really slow and allows just one request at a time, therefore it was not possible to calculate the model for 1ltz with the adjusted alignment in time. == Evaluation of the calculated models == === Selection of the reference structures === We had the following choice of reference structures for [http://www.uniprot.org/uniprot/P00439#section_x-ref PAH]: {| border="1" |- ! Entry ! Method ! Resolution (A) ! Chain ! Positions |- | 1DMW | X-Ray | 2.00 | A | 118-424 |- | 1J8T | X-Ray | 1.70 | A | 103-427 |- | 1J8U | X-Ray | 1.50 | A | 103-427 |- | 1KW0 | X-Ray | 2.50 | A | 103-427 |- | 1LRM | X-Ray | 2.10 | A | 103-427 |- | 1MMK | X-Ray | 2.00 | A | 103-427 |- | 1MMT | X-Ray | 2.00 | A | 103-427 |- | 1PAH | X-Ray | 2.00 | A | 117-424 |- | 1TDW | X-Ray | 2.10 | A | 117-424 |- | 1TG2 | X-Ray | 2.20 | A | 117-424 |- | 2PAH | X-Ray | 3.10 | A/B | 118-452 |- | 3PAH | X-Ray | 2.00 | A | 117-424 |- | 4PAH | X-Ray | 2.00 | A | 117-424 |- | 5PAH | X-Ray | 2.10 | A | 117-424 |- | 6PAH | X-Ray | 2.15 | A | 117-424 |} All these structures have in common that they did not solve the structure of the whole PAH protein. In addition, there is no complete true apo structure available either. All structures have at least a Fe2+ atom bound. So we defined these structures as our apo structure. Finally, we decided to select 1J8T (apo) and 1J8U (complexed). As mentioned before our apo structure has complexed Fe2+ and our complexed structure is complexed with Fe2+ and BH4 (5,6,7,8-TETRAHYDROBIOPTERIN). The reason for our decision was that both structures are solved from the same group which somehow guaranties a more consistent methodology as if we had selected structures from two different groups. Another reason is the resolution, both structures are the two with the best resolved resolution which is 1.5 Angstrom and 1.7 Angstrom for 1J8U and 1J8T respectively. Finally for more easy comparison, both structures include the same range of amino acids which is from 103 to 427. === Numeric evaluation of the calculated models === ==== Modeller ==== ===== Description of the quantitative Modeller scores ===== '''molpdf (molecular PDF):''' This measure is the sum of all restrains and is the standard score of Modeller. This score is not absolute which means it can be only used for ranking models from the same alignment. Lower means better for this score. (Source:[http://salilab.org/modeller/tutorial/cryoem/assess.html] ) '''DOPE (Discrete Optimized Protein Energy):''' "this statistical potential is based on an improved reference state that corresponds to non interacting atoms in a homogeneous sphere with the radius dependent on a sample native structure; it thus accounts for the finite and spherical shape of the native structures". It is an absolute score. Lower means better for this score. (Source: [http://en.wikipedia.org/wiki/Discrete_optimized_protein_energy]) '''GA341:''' "The method uses the percentage sequence identity between the template and the model as a parameter." The range is from 0 to 1. Higher means better. However, this method is not as good as the other described methods to judge the quality of a model. (Source: [http://salilab.org/modeller/manual/node195.html] and [http://salilab.org/modeller/tutorial/cryoem/assess.html]) ===== Values ===== We have chosen three scores to be calculated by Modeller: molpdf, DOPE and GA341 {| border="1" ! model ! molpdf ! DOPE ! GA341 |- | 1phz | 2568.91309 | -53912.11328 | 1.00000 |- | 1toh | 20481.25977 | -37609.82031 | 1.00000 |- | 1ltz | 6824.36182 | -37422.13672 | 0.98868 |- | 1phz with adjusted alignment | 2567.52441 | -49139.31250 | 1.00000 |- | 1toh with adjusted alignment | 1790.78723 | -38113.64453 | 1.00000 |- | 1ltz with adjusted alignment | 1197.55518 | -26429.44531 | 1.00000 |- | multiple alignment | 61561.77344 | -29682.17578 | 0.00012 |} ==== Swissmodel ==== ===== Automated Mode: Modelling with template structure 1phz_A (>60%) ===== '''QMEAN Z-Score:''' -0.828 '''QMEAN4 global scores:''' {| border="1" |- ! QMEANscore4 ! Estimated absolute model quality ! Score components |- | 0.715 | [[File:QMEAN plots 1phz template.pdb plot.png | 300px]] [[File:QMEAN plots 1phz template pdb plot.png density plot.png | 300px]] | [[File:QMEAN plots 1phz template plot.png slider.png | 300px]] |} '''Local scores:''' {| border="1" |- ! Coloring by residue error ! Residue error plot |- | [[File:1phz template coloring by residue error.jpeg | 300px]] | [[File:QMEAN plots energy profile plots 1phz template.pdb local energy profile QMEANlocal.png | 300px]] |} '''Global scores: QMEAN4:''' {| border="1" ! Scoring function term ! Raw score ! Z-score |- | C_beta interaction energy | -157.98 | -0.16 |- | All-atom pairwise energy | -12503.57 | -0.1 |- | Solvation energy | -50.66 | 1.01 |- | Torsion angle energy | -78.77 | -1.48 |- | QMEAN4 score | 0.715 | -0.83 |} '''Local Model Quality Estimation: Anolea / QMEAN / Gromos:''' [[File:Local quality estimation 1phz template annolea qmean gromos.png | 300px]] ===== Automated Mode: Modelling with template structure 1toh_A (>40%) ===== '''QMEAN Z-Score:''' -2.745 '''QMEAN4 global scores:''' {| border="1" |- ! QMEANscore4 ! Estimated absolute model quality ! Score components |- | 0.604 | [[File:1toh QMEAN plots Batch.1.short.pdb plot.png | 300px]] [[File:1toh QMEAN plots Batch.1.short.pdb plot.png density plot.png | 300px]] | [[File:1toh QMEAN plots Batch.1.short.pdb plot.png slider.png | 300px]] |} '''Local scores:''' {| border="1" |- ! Coloring by residue error ! Residue error plot |- | [[File:1toh residue error structure.jpeg | 300px]] | [[File:1toh QMEAN plots energy profile plots Batch.1.short.pdb local energy profile QMEANlocal.png | 300px]] |} '''Global scores: QMEAN4:''' {| border="1" ! Scoring function term ! Raw score ! Z-score |- | C_beta interaction energy | -78.67 | -1.16 |- | All-atom pairwise energy | -7899.47 | -0.89 |- | Solvation energy | -20.56 | -1.3 |- | Torsion angle energy | -47.34 | -2.22 |- | QMEAN4 score | 0.604 | -2.74 |} '''Local Model Quality Estimation: Anolea / QMEAN / Gromos:''' [[File:1toh local mode quality estimation anolea qmean gromos.png | 300px]] ===== Automated Mode: Modelling with template structure 1ltz_A (<40%) ===== '''QMEAN Z-Score:''' -4.282 '''QMEAN4 global scores:''' {| border="1" |- ! QMEANscore4 ! Estimated absolute model quality ! Score components |- | 0.47 | [[File:1ltz QMEAN plots Batch.1.short.pdb plot.png | 300px]] [[File:1 ltz QMEAN plots Batch.1.short.pdb plot.png density plot.png | 300px]] | [[File:1 ltz QMEAN plots Batch.1.short.pdb plot.png slider.png | 300px]] |} '''Local scores:''' {| border="1" |- ! Coloring by residue error ! Residue error plot |- | [[File:1ltz residue error pdb plot.jpeg | 300px]] | [[File:1ltz QMEAN plots energy profile plots Batch.1.short.pdb local energy profile QMEANlocal.png | 300px]] |} '''Global scores: QMEAN4:''' {| border="1" ! Scoring function term ! Raw score ! Z-score |- | C_beta interaction energy | -40.25 | -2.1 |- | All-atom pairwise energy | -3528.81 | -2.29 |- | Solvation energy | -15.22 | -1.18 |- | Torsion angle energy | -4.99 | -3.78 |- | QMEAN4 score | 0.47 | -4.28 |} '''Local Model Quality Estimation: Anolea / QMEAN / Gromos:''' [[File:1ltz local model quality estimation anolea qmean gromos.png | 300px]] ===== Alignment Mode: Modeling with adjusted alignment 1phz_A (>60%) ===== '''QMEAN Z-Score:''' -2.786 '''QMEAN4 global scores:''' {| border="1" |- ! QMEANscore4 ! Estimated absolute model quality ! Score components |- | 0.6 | [[File:QMEAN plots 1phz imp template.pdb plot.png | 300px]] [[File:QMEAN plots 1phz imp template pdb plot.png density plot.png | 300px]] | [[File:QMEAN plots 1phz imp template plot.png slider.png | 300px]] |} '''Local scores:''' {| border="1" |- ! Coloring by residue error ! Residue error plot |- | [[File:1phz imp template coloring by residue error.jpeg | 300px]] | [[File:QMEAN plots energy profile plots 1phz imp template.pdb local energy profile QMEANlocal.png | 300px]] |} '''Global scores: QMEAN4:''' {| border="1" ! Scoring function term ! Raw score ! Z-score |- | C_beta interaction energy | -87.10 | -1.26 |- | All-atom pairwise energy | -8126.09 | -1.51 |- | Solvation energy | -29.98 | -0.84 |- | Torsion angle energy | -58.30 | -2.39 |- | QMEAN4 score | 0.600 | -2.79 |} '''Local Model Quality Estimation: Anolea / QMEAN / Gromos:''' [[File:Local quality estimation 1phz imp template annolea qmean gromos.png | 300px]] ===== Alignment Mode: Modeling with adjusted alignment 1toh_A (>40%) ===== '''QMEAN Z-Score:''' -4.498 '''QMEAN4 global scores:''' {| border="1" |- ! QMEANscore4 ! Estimated absolute model quality ! Score components |- | 0.494 | [[File:QMEAN plots 1toh imp template.pdb plot.png | 300px]] [[File:QMEAN plots 1toh imp template pdb plot.png density plot.png | 300px]] | [[File:QMEAN plots 1toh imp template plot.png slider.png | 300px]] |} '''Local scores:''' {| border="1" |- ! Coloring by residue error ! Residue error plot |- | [[File:1toh imp template coloring by residue error.jpeg| 300px]] | [[File:QMEAN plots energy profile plots 1toh imp template.pdb local energy profile QMEANlocal.png| 300px]] |} '''Global scores: QMEAN4:''' {| border="1" ! Scoring function term ! Raw score ! Z-score |- | C_beta interaction energy | -21.70 | -2.19 |- | All-atom pairwise energy | -4082.14 | -2.32 |- | Solvation energy | -3.04 | -3.04 |- | Torsion angle energy | -34.43 | -2.83 |- | QMEAN4 score | 0.494 | -4.50 |} '''Local Model Quality Estimation: Anolea / QMEAN / Gromos:''' [[File:Local quality estimation 1toh imp template annolea qmean gromos.png | 300px]] ===== Alignment Mode: Modeling with adjusted alignment 1ltz_A (<40%) ===== '''QMEAN Z-Score:''' -4.673 '''QMEAN4 global scores:''' {| border="1" |- ! QMEANscore4 ! Estimated absolute model quality ! Score components |- | 0.447 | [[File:QMEAN plots 1ltz imp template.pdb plot.png | 300px]] [[File:QMEAN plots 1ltz imp template pdb plot.png density plot.png | 300px]] | [[File:QMEAN plots 1ltz imp template plot.png slider.png | 300px]] |} '''Local scores:''' {| border="1" |- ! Coloring by residue error ! Residue error plot |- | [[File:1ltz imp template coloring by residue error.jpeg | 300px]] | [[File:QMEAN plots energy profile plots 1ltz imp template.pdb local energy profile QMEANlocal.png | 300px]] |} '''Global scores: QMEAN4:''' {| border="1" ! Scoring function term ! Raw score ! Z-score |- | C_beta interaction energy | -18.68 | -2.69 |- | All-atom pairwise energy | -1884.30 | -2.99 |- | Solvation energy | -9.58 | -1.72 |- | Torsion angle energy | -7.34 | -3.55 |- | QMEAN4 score | 0.447 | -4.67 |} '''Local Model Quality Estimation: Anolea / QMEAN / Gromos:''' [[File:Local quality estimation 1ltz imp template annolea qmean gromos.png | 300px]] ==== iTasser ==== {| border="1" | Model | C-Score | estimated TM-score | estimated RMSD |- | 1phz | 0.150 | 0.73±0.11 | 6.8±4.0Å |- | 1toh | 0.074 | 0.72±0.11 | 6.9±4.1Å |- | 1ltz | -0.380 | 0.66±0.13 | 7.9±4.4Å |- | 1phz adjusted | 1.650 | 0.95±0.05 | 3.5±2.4Å |- | 1toh adjusted | 1.944 | 0.99±0.04 | 2.6±1.9Å |- | 1ltz adjusted | 2.053 | 0.99±0.04 | 1.7±1.5Å |} ====3D-JigSaw==== =====1phz - sequence identity > 60 % ===== [[File:Pah jig 1phz Energy.gif]] {| border="1" ! MODEL ! ENERGY ! COVERAGE ! START ! END ! RAMACHANDRAN PLOT |- | MODEL_1 | -638.18 | 1.00 | 1 | 452 | [[File:Pah jig 1phz rmp model 1.png|300px]] |- | MODEL_2 | -637.27 | 1.00 | 1 | 452 | [[File:Pah jig 1phz rmp model 2.png|300px]] |- | MODEL_3 | -634.89 | 1.00 | 1 | 452 | [[File:Pah jig 1phz rmp model 3.png|300px]] |- | MODEL_4 | -634.77 | 0.96 | 19 | 452 | [[File:Pah jig 1phz rmp model 4.png|300px]] |- | MODEL_5 | -632.78 | 1.00 | 1 | 452 | [[File:Pah jig 1phz rmp model 5.png|300px]] |} =====1toh - sequence identity > 40 % ===== [[File:pah_jig_1toh_Energy.gif]] {| border="1" ! MODEL ! ENERGY ! COVERAGE ! START ! END ! RAMACHANDRAN PLOT |- | MODEL_1 | -557.84 | 1.00 | 1 | 452 | [[File:pah_jig_1toh_rmp_model_1.png|300px]] |- | MODEL_2 | -557.83 | 1.00 | 1 | 452 | [[File:pah_jig_1toh_rmp_model_2.png|300px]] |- | MODEL_3 | -556.11 | 1.00 | 1 | 451 | [[File:pah_jig_1toh_rmp_model_3.png|300px]] |- | MODEL_4 | -548.57 | 1.00 | 1 | 452 | [[File:pah_jig_1toh_rmp_model_4.png|300px]] |- | MODEL_5 | -547.22 | 1.00 | 1 | 452 | [[File:pah_jig_1toh_rmp_model_5.png|300px]] |} =====1ltz - sequence identity < 40 % ===== [[File:pah_jig_1ltz_Energy.gif]] {|border="1" ! MODEL ! ENERGY ! COVERAGE ! START ! END ! RAMACHANDRAN PLOT |- | MODEL_1 | -492.57 | 1.00 | 1 | 452 | [[File:Pah jig 1ltz rmp model 1.png|300px]] |- | MODEL_2 | -490.72 | 1.00 | 1 | 452 | [[File:Pah jig 1ltz rmp model 2.png|300px]] |- | MODEL_3 | -489.81 | 1.00 | 1 | 452 | [[File:Pah jig 1ltz rmp model 3.png|300px]] |- | MODEL_4 | -489.75 | 1.00 | 1 | 452 | [[File:Pah jig 1ltz rmp model 4.png|300px]] |- | MODEL_5 | -489.73 | 1.00 | 1 | 452 | [[File:Pah jig 1ltz rmp model 5.png|300px]] |} === Comparison to experimental structure === To calculate the C-alpha RMSD we used [http://www.ebi.ac.uk/Tools/dalilite/index.html? DaliLite]. Later we changed to the command line tool <code>sap file1.pdb file2.pdb</code>. To calculate the TM-Score we used the [http://zhanglab.ccmb.med.umich.edu/TM-score/ TM-score webservice] from the University of Michigan alternative. Later we changed to the command line tool <code>TMS model.pdb native.pdb</code>. To calculate the RMSD of the 6A radius of the catalytic center we had to first identify the catalytic center. We defined the center position of the catalytic side as the position where our Fe2+ atom is. With the position in hand we now have to extract the residues in a 6A radius around this Fe2+ atom. In order to do so we executed the following steps: * We opened the complexed or apo structure and one of the modeled structures with Pymol. * Then we aligned both structures to each other * Then we selected the Fe2 atom of the apo/complexed structure and expanded this selection by 6A, residue * Then we extracted the selected residues into two objects each object contains only the residues of either the apo/complexed structure or the modeled structure * Then we saved both objects in seperate PDB structures * Now we used the [http://blue11.bch.msu.edu/mmtsb/rms.pl rms.pl script] to calculate the all atom RMSD with the following command "./rms.pl -out all first.pdb second.pdb". This script is already installed in /apps/bin and can be called in the commandline by rms.pl. We had a lot of models, therefore it was useful to write a program to do the evaluation automatically ([[pah_hom_autom_eval|sourcecode]]). ==== Modeller ==== ===== Standard Workflow ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | 1PHZ Chain: A | 1J8T | Apo | 0.9 | 0.9711 | 0.276 |- | 1PHZ Chain: A | 1J8U | Complexed | 0.9 | 0.6494 | 0.271 |- | 1TOH Chain: A | 1J8T | Apo | 1.9 | 0.6502 | 0.282 |- | 1TOH Chain: A | 1J8U | Complexed | 1.9 | 0.8850 | 0.242 |- | 1LTZ Chain: A | 1J8T | Apo | 2.3 | 0.6986 | 1.638 |- | 1LTZ Chain: A | 1J8U | Complexed | 2.3 | 0.6985 | 1.581 |- | Multiple | 1J8T | Apo | 2.1 | 0.1886 | 3.814 |- | Multiple | 1J8U | Complexed | 2.1 | 0.1879 | 3.809 |} ===== Adjusted Alignment Workflow ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | 1PHZ Chain: A | 1J8T | Apo | 1.0 | 0.1957 | 0.298 |- | 1PHZ Chain: A | 1J8U | Complexed | 1.0 | 0.1954 | 0.310 |- | 1TOH Chain: A | 1J8T | Apo | 1,1 | 0.1592 | 0.276 |- | 1TOH Chain: A | 1J8U | Complexed | 1.1 | 0.1592 | 0.328 |- | 1LTZ Chain: A | 1J8T | Apo | 1.7 | 0.1021 | 3.562 |- | 1LTZ Chain: A | 1J8U | Complexed | 1.7 | 0.1020 | 0.832 |} ==== Swissmodel ==== ===== Standard Workflow ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | 1PHZ Chain: A | 1J8T | Apo | 0.9 | 0.7400 | 0.5162 |- | 1PHZ Chain: A | 1J8U | Complexed | 0.9 | 0.7408 | 0.5154 |- | 1TOH Chain: A | 1J8T | Apo | 1.3 | 0.8889 | 0.4616 |- | 1TOH Chain: A | 1J8U | Complexed | 1.2 | 0.8894 | 0.3361 |- | 1LTZ Chain: A | 1J8T | Apo | 2.3 | 0.8816 | 0.9225 |- | 1LTZ Chain: A | 1J8U | Complexed | 2.3 | 0.8814 | 0.9208 |} ===== Adjusted Alignment Workflow ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | 1PHZ Chain: A | 1J8T | Apo | 0.645 | 0.0880 | 0.355 |- | 1PHZ Chain: A | 1J8U | Complexed | 0.615 | 0.0878 | 0.267 |- | 1TOH Chain: A | 1J8T | Apo | 0.996 | 0.0894 | 0.227 |- | 1TOH Chain: A | 1J8U | Complexed | 0.993 | 0.0894 | 0.262 |- | 1LTZ Chain: A | 1J8T | Apo | 1.542 | 0.0782 | 1.204 |- | 1LTZ Chain: A | 1J8U | Complexed | 1.542 | 0.0790 | 3.839 |} ==== iTasser ==== ===== Standard Workflow ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | 1PHZ Chain: A | 1J8T | Apo | 0.722 | 0.6596 | 0.241 |- | 1PHZ Chain: A | 1J8U | Complexed | 0.698 | 0.6604 | 0.279 |- | 1TOH Chain: A | 1J8T | Apo | 0.749 | 0.6571 | 0.299 |- | 1TOH Chain: A | 1J8U | Complexed | 0.731 | 0.6578 | 0.297 |- | 1LTZ Chain: A | 1J8T | Apo | 0.644 | 0.6606 | 0.374 |- | 1LTZ Chain: A | 1J8U | Complexed | 0.610 | 0.6620 | 0.351 |} ===== Adjusted Alignment Workflow ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | 1PHZ Chain: A | 1J8T | Apo | 0.829 | 0.0903 | 0.424 |- | 1PHZ Chain: A | 1J8U | Complexed | 0.816 | 0.0909 | 0.441 |- | 1TOH Chain: A | 1J8T | Apo | 0.720 | 0.0875 | 0.449 |- | 1TOH Chain: A | 1J8U | Complexed | 0.671 | 0.0873 | 0.416 |- | 1LTZ Chain: A | 1J8T | Apo | 1.382 | 0.0776 | 0.700 |- | 1LTZ Chain: A | 1J8U | Complexed | 1.372 | 0.0775 | 0.851 |} ==== 3D JigSaw ==== ===== 1phz ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | Model 1 | 1J8T | Apo | 1.121 | 0.6466 | 0.237 |- | Model 1 | 1J8U | Complexed | 1.106 | 0.6475 | 0.439 |- | Model 2 | 1J8T | Apo | 1.055 | 0.6422 | 0.265 |- | Model 2 | 1J8U | Complexed | 1.040 | 0.6428 | 0.310 |- | Model 3 | 1J8T | Apo | 1.035 | 0.6340 | 0.261 |- | Model 3 | 1J8U | Complexed | 1.025 | 0.6347 | 0.277 |- | Model 4 | 1J8T | Apo | 1.069 | 0.6664 | 0.450 |- | Model 4 | 1J8U | Complexed | 1.063 | 0.6670 | 0.512 |- | Model 5 | 1J8T | Apo | 1.055 | 0.6422 | 0.251 |- | Model 5 | 1J8U | Complexed | 1.040 | 0.6428 | 0.369 |} ===== 1toh ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | Model 1 | 1J8T | Apo | 5.443 | 0.5090 | 0.734 |- | Model 1 | 1J8U | Complexed | 5.453 | 0.5095 | 0.766 |- | Model 2 | 1J8T | Apo | 5.443 | 0.5090 | 0.734 |- | Model 2 | 1J8U | Complexed | 5.453 | 0.5095 | 0.766 |- | Model 3 | 1J8T | Apo | 4.832 | 0.4450 | 1.879 |- | Model 3 | 1J8U | Complexed | 4.838 | 0.4456 | 0.276 |- | Model 4 | 1J8T | Apo | 3.262 | 0.5717 | 0.432 |- | Model 4 | 1J8U | Complexed | 3.258 | 0.5722 | 0.453 |- | Model 5 | 1J8T | Apo | 3.137 | 0.5708 | 0.432 |- | Model 5 | 1J8U | Complexed | 3.128 | 0.5715 | 0.453 |} ===== 1ltz ===== {| border="1" |- ! Template Structure ! Compared To ! Apo/Complexed ! C-alpha RMSD ! TM score ! All Atoms RMSD, 6A |- | Model 1 | 1J8T | Apo | 2.538 | 0.6247 | 0.344 |- | Model 1 | 1J8U | Complexed | 2.528 | 0.6255 | 0.361 |- | Model 2 | 1J8T | Apo | 2.540 | 0.6244 | 0.358 |- | Model 2 | 1J8U | Complexed | 2.531 | 0.6255 | 0.385 |- | Model 3 | 1J8T | Apo | 2.540 | 0.6244 | 0.358 |- | Model 3 | 1J8U | Complexed | 2.531 | 0.6255 | 0.384 |- | Model 4 | 1J8T | Apo | 2.541 | 0.6244 | 0.349 |- | Model 4 | 1J8U | Complexed | 2.531 | 0.6255 | 0.377 |- | Model 5 | 1J8T | Apo | 2.540 | 0.6244 | 0.349 |- | Model 5 | 1J8U | Complexed | 2.531 | 0.6255 | 0.377 |} == Discussion == [[Image:Pah_multi_modeller.png|thumb|top|Figure 3: image of the model of modeller with multiple targets]] Our chosen templates were not that easy for homology modelling. Phenylalanine hydroxylase contains two domains Biopterin_H and ACT. Only 1phz contains both domains. All three templates have large gaps in the Biopterin_H domain, especially in 1ltz the first part of the domain seems to be missing. Modeller was the fastest method and relatively easy to handle. But it has several drawbacks. The alignment by Modeller is sometimes far from good. For example the Biopterin_H domain of 1ltz was splitted by Modeller. This is also reflected by the low TM-Scores of 1ltz and 1toh. But it was also relatively easy to improve the alignment, such that the alignments are much more similar to the family prediction by PFAM. For 1phz the alignment was almost perfect and only small adjustments were necessary. For the other two templates we deleted large parts at the beginning and at the end of the target, which were not covered by the templates. Most modeling methods tend to do nonsense especially at the beginning and end of a model, if there is no information given by the template (one kind of possible nonsense is shown in Figure 3). All three adjusted alignments are shorter than the target sequence. Therefore the TM-score in the experimental validation is not reliable, because it is dependent of the model length. Regarding the RMSD and the RMSD of the residues around the Fe in the native structures, the adjustment of the alignments was at least for 1ltz and 1toh very successful. In our opinion the adjustment of an alignment is almost always wise, but it is especially crucial for templates with low sequence identity. We tried to use the automated workflow of Modeller with multiple templates. Even with templates with high sequence identity to the target, the created model was obviously not reasonable. That is why we did not use any of these multiple template models. Swissmodel was run in the server version. The modeling took less than ten minutes. Regarding the RMSD and the RMSD of the residues around the Fe in the native structures the usage of the adjusted alignments seems to improve the modeling. It surprised that Swissmodel performed better for the low identity templates than Modeller. But created for 1phz a model worse than that of Modeller. Perhaps Swissmodel does to much in the automatic workflow and loses information of templates with high sequence identity. iTasser ran for about 2 days per model. Users are only allowed to upload one request at a time. Therefore iTasser was no fun. iTasser seems to do a lot of stuff. Therefore the models were worse with our adjusted alignments. But over all the models of the automated iTasser workflow are not that good. Adjusted models of Modeller and SwissModel are better in the RMSD and Fe-RMSD. The TM-Score is always below the automated workflow models of Modeller and SwissModel. This result was surprising, because iTasser is known from the CASP contest to be at the top of the automated prediction servers. Perhaps simple homology modeling and manually curated alignments are more powerful than these servers. The results of 3D-jigsaw are quite disappointing. It looks as if 3d-jigsaw was too much influenced by the iTasser models. Perhaps with another set of models 3d-jigsaw would be more reasonable. But at least it was not able to distinguish the "good" models of the "bad" models in the sets and therefore it is doubtful that in praxis it would perform better. Users usually do not know how good or bad their models are and that is why they use such a server. The ranking of the chosen models is not really analyzable, because all the produced models are almost of the same quality.

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