Difference between revisions of "Task 4: Homology based structure predictions"

From Bioinformatikpedia
(→‎Comparison to experimental structure)
(→‎Swissmodel)
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[[File:1ltz local model quality estimation anolea qmean gromos.png | 300px]]
  
[[File:1ltz local model quality estimation anolea qmean gromos.png | 300px]]
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===== Alignment Mode: Modeling with adjusted alignment 1phz_A (>60%) =====
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'''QMEAN Z-Score:''' -2.786
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'''QMEAN4 global scores:'''
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{| border="1"
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|-
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! QMEANscore4
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! Estimated absolute model quality
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! Score components
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|-
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| 0.6
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| [[File:QMEAN plots 1phz imp template.pdb plot.png | 300px]] [[File:QMEAN plots 1phz imp template pdb plot.png density plot.png | 300px]]
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| [[File:QMEAN plots 1phz imp template plot.png slider.png | 300px]]
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|}
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'''Local scores:'''
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{| border="1"
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|-
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! Coloring by residue error
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! Residue error plot
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|-
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| [[File:1phz imp template coloring by residue error.jpeg | 300px]]
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| [[File:QMEAN plots energy profile plots 1phz imp template.pdb local energy profile QMEANlocal.png | 300px]]
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|}
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'''Global scores: QMEAN4:'''
  +
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{| border="1"
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! Scoring function term
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! Raw score
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! Z-score
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|-
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| C_beta interaction energy
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| -87.10
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| -1.26
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|-
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| All-atom pairwise energy
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| -8126.09
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| -1.51
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|-
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| Solvation energy
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| -29.98
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| -0.84
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|-
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| Torsion angle energy
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| -58.30
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| -2.39
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|-
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| QMEAN4 score
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| 0.600
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| -2.79
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|}
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'''Local Model Quality Estimation: Anolea / QMEAN / Gromos:'''
  +
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[[File:Local quality estimation 1phz imp template annolea qmean gromos.png | 300px]]
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===== Alignment Mode: Modeling with adjusted alignment 1toh_A (>40%) =====
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  +
'''QMEAN Z-Score:''' -4.498
  +
  +
'''QMEAN4 global scores:'''
  +
  +
{| border="1"
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|-
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! QMEANscore4
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! Estimated absolute model quality
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! Score components
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|-
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| 0.494
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| [[File:1toh imp QMEAN plots Batch.1.short.pdb plot.png | 300px]] [[File:1toh imp QMEAN plots Batch.1.short.pdb plot.png density plot.png | 300px]]
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| [[File:1toh imp QMEAN plots Batch.1.short.pdb plot.png slider.png | 300px]]
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|}
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  +
  +
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'''Local scores:'''
  +
  +
{| border="1"
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|-
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! Coloring by residue error
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! Residue error plot
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|-
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| [[File:1toh imp residue error structure.jpeg | 300px]]
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| [[File:1toh imp QMEAN plots energy profile plots Batch.1.short.pdb local energy profile QMEANlocal.png | 300px]]
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|}
  +
  +
  +
  +
'''Global scores: QMEAN4:'''
  +
  +
{| border="1"
  +
! Scoring function term
  +
! Raw score
  +
! Z-score
  +
|-
  +
| C_beta interaction energy
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| -21.70
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| -2.19
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|-
  +
| All-atom pairwise energy
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| -4082.14
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| -2.32
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|-
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| Solvation energy
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| -3.04
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| -3.04
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|-
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| Torsion angle energy
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| -34.43
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| -2.83
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|-
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| QMEAN4 score
  +
| 0.494
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| -4.50
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|}
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  +
  +
  +
  +
'''Local Model Quality Estimation: Anolea / QMEAN / Gromos:'''
  +
  +
[[File:1toh imp local mode quality estimation anolea qmean gromos.png | 300px]]
  +
  +
  +
  +
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===== 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
  +
|-
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| 0.447
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| [[File:1ltz imp QMEAN plots Batch.1.short.pdb plot.png | 300px]] [[File:1 ltz imp QMEAN plots Batch.1.short.pdb plot.png density plot.png | 300px]]
  +
| [[File:1ltz imp QMEAN plots Batch.1.short.pdb plot.png slider.png | 300px]]
  +
|}
  +
  +
  +
  +
'''Local scores:'''
  +
  +
{| border="1"
  +
|-
  +
! Coloring by residue error
  +
! Residue error plot
  +
|-
  +
| [[File:1ltz imp residue error pdb plot.jpeg | 300px]]
  +
| [[File:1ltz imp 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
  +
| -18.68
  +
| -2.69
  +
|-
  +
| All-atom pairwise energy
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| -1884.30
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| -2.99
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|-
  +
| Solvation energy
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| -9.58
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| -1.72
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|-
  +
| Torsion angle energy
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| -7.34
  +
| -3.55
  +
|-
  +
| QMEAN4 score
  +
| 0.447
  +
| -4.67
  +
|}
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  +
'''Local Model Quality Estimation: Anolea / QMEAN / Gromos:'''
  +
  +
[[File:1ltz imp local model quality estimation anolea qmean gromos.png | 300px]]
   
 
==== iTasser ====
  
==== iTasser ====

Revision as of 23:17, 10 June 2011

Task description

The full description of this task can be found 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:

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%


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.

Seeds by PFAM - ACT:

REFERENCE SLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKkDEYEFFTHLD-KRSL
1PHZ SLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNkDEYEFFTYL------

Seeds by PFAM - Biopterin_H

REFERENCE PWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCL-SEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQK
1PHZ PWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCL-SDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT-----------------------
1TOH PWFPRKVSELDKC-----------DLDHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASD-EEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSL-SEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDELHTLAHALS-
1LTZ ------------------------------------------------------PQPLDRYSAEDHATWATLYQRQCKLLPGRACDEFLEGLERLEV----DADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKAlGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLdSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQ--------------------------------------------------------

unadjusted alignment of 1TOH

REFERENCE MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1TOH -------------K-------------------------------------------------------------------------------------------------------VPWFPRKVSELDKCD---L--------DHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASDEEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSLSEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDELHTLAHALSAIS
PSI -------------C-------------------------------------------------------------------------------------------------------CCCCCCCCCCCCCCC---C--------CCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHCCCEEEECCCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHCEEEEEEEEEEEEECCCCEECCCCCCCCCCHHCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCEEEEECCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCEECCCCHHHHHHHHHHHHHHHHHHHHHHHHHC

unadjusted alignment of 1PHZ

REFERENCE MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1PHZ ------------------GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFFTYLDKRTKPVLGSIIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCLSDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT-------------------------
PSI ------------------CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEEEEECCCCCCCHHHHHHHHHCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHHHHHHCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCCEEEECCCCCCHHHHHHHHHCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHHHEEEEEEEEEEEECCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHHHHHHHCCCCCCCCCCCCCCEEEECCCC-------------------------

unadjusted alignment of 1LTZ

REFERENCE MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCL-SEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCH HHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHH CCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1LTZ -----------------F--------------------------V-----V-------------------------------------PDITT-----RKNVG-----LSHDANDFTLP------QPLDR-------YSA-----------------------------------------EDHATWATLYQRQCKLLPGRACDEFLEGLERLE----VDADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKALGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLDSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQL------FDAD----FAPLY------LQLAD-AQPWG--AGDIAP------DDL--VL
PSI -----------------C--------------------------C-----C-------------------------------------CCCCC-----CCCCC-----CCCCCCCCCCC------CCCCC-------CCH-----------------------------------------HHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCC----CCCCCCCCCHHHHHHHHHHHCCEEEECCCCCCHHHHHHHHHCCCCCEEECCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCCCCHHHHHHHHHEEEEEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCCCEEECCCHHHHHCCCCCCCCCCCCEEEECCHHHH------HHHC----CHHHH------HHHHH-CCCCC--CCCCCC------CCC--CC

adjusted alignment of 1LTZ

REFERENCE PIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAAT
PSI CCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHH
1LTZ PQPLDRYSAEDHATWATLYQRQCKLLPGRACDEFLEGLERLEV----DADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKAlGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLdSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQLFDADFAPL
PSI CCCCCCCCHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHHHCCEEEECCCCCCHHHHHHHHHCCCCCEEECCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCCCCHHHHHHHHHEEEEEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCCCEEECCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHCCHHHHHHH

adjusted alignment of 1PHZ

REFERENCE GQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQ
PSI CCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHH
1PHZ GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNkDEYEFFTYLDKRTKPVLGSIIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCL-SDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT
PSI CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEEEEECCCCCCCHHHHHHHHHCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHHHHHHCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCCEEEECCCCCCHHHHHHHHHCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCH-HHHHHHHHHEEEEEEEEEEEECCCCEEECCCCCCCCCCCCCCC-CCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHHHHHHHCCCCCCCCCCCCCCEEEECCCC

adjusted alignment of 1TOH

REFERENCE VPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI CCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1TOH VPWFPRKVSELDKC-----------DLDHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASD-EEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSL-SEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDELHTLAHALSA
PSI CCCCCCCCCCCCCC CCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHCCCEEEECCCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHCEEEEEEEEEEEEECCCCEECCCCCCCCCCHHCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCEEEEECCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCEECCCCHHHHHHHHHHHHHHHHHHHHHHHHHC

Homology modelling with Modeller

Modeller uses two types of files to be run. The first one contains the used alignment in the PIR-format (see PIR FORMAT), the second one is a python script, which tells Modeller which steps it has to perform. There are some examples in /apps/modeller9.9/examples/automodel. But python seems to be falsely configured on the virtual machines (at least the linux virtual machine).

  • The used fix of the python installation is described at the 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 software section.

After all this stuff we tried to write a alignment-file with the hhsearch alignments and a python-file (according to the example /apps/modeller9.9/examples/automodel/model-default.py. But modeller seems to be very sensible due to missing acids in the coordinate section of the pdb, which are of course mentioned in the sequence used in the alignment.

To avoid this problem there are at least two possibilities:

  • Repair the pdb by the script repairPDB. Map the sequence in the alignment on the sequence in the coordinate section of the used pdb (a lot of work - need some kind of alignment and a pdb-parser... both is not trivial)
  • The other possibility is to let Modeller create the alignment on the basis of the repaired PDB (with repairPDB).

We have chosen the second option, which is really nice described in the Modeller tutorial (see basic modeller tutorial).

At the example of 1PHZ. We prepared three files.

  • The alignment file, which contains only the reference sequence: pah.ali
    >P1;PAH
    sequence:reference::::::::
    MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEEN
    DVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKD
    TVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPI
    PRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQ
    FLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGH
    VPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLL
    SSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATI
    PRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK*
  • The python script, which tells Modeller to create an alignment
    from modeller import *

    env = environ()
    aln = alignment(env)
    mdl = model(env, file='../structures/1phz.pdb', model_segment=('FIRST:A','LAST:A'))
    aln.append_model(mdl, align_codes='1phz', atom_files='../structures/1phz.pdb')
    aln.append(file='pah.ali', align_codes='PAH')
    aln.align2d()
    aln.write(file='phz.ali', alignment_format='PIR')

  • Then we tried to optimize the alignment. (for the different criteria see below)
  • The python script, which tells Modeller to create a model
    from modeller.automodel import *

    a = automodel(env, alnfile='phz.ali',
    knowns='1phz', sequence='PAH',
    assess_methods=(assess.DOPE, assess.GA341))
    a.starting_model = 1
    a.ending_model = 1
    a.make()

Homology modelling 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:

Category Template Target Image
> 60% sequence identity 1PHZ Chain: A P00439 (Phenylalanine-4-hydroxylase) 1phz A template auto model.png
> 40% sequence identity 1TOH Chain: A P00439 (Phenylalanine-4-hydroxylase) 1toh A template auto model.png
< 40% sequence identity 1LTZ Chain: A P00439 (Phenylalanine-4-hydroxylase) 1ltz A template auto model.png

Homology modelling with iTasser

ITasser is a prediction server, which participated in several CASP competitions. It claims of itself to be the best one. The interface of this server can be seen in Figure 1. 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.

Evaluation of the calculated models

Selection of the reference structures

We had the following choice of reference structures for PAH:

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

We have chosen three scores to be calculated by Modeller: molpdf, DOPE and GA341

model modlpdf 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
1loh 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:

QMEANscore4 Estimated absolute model quality Score components
0.715 QMEAN plots 1phz template.pdb plot.png QMEAN plots 1phz template pdb plot.png density plot.png QMEAN plots 1phz template plot.png slider.png


Local scores:

Coloring by residue error Residue error plot
1phz template coloring by residue error.jpeg QMEAN plots energy profile plots 1phz template.pdb local energy profile QMEANlocal.png


Global scores: QMEAN4:

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:

Local quality estimation 1phz template annolea qmean gromos.png


Automated Mode: Modelling with template structure 1toh_A (>40%)

QMEAN Z-Score: -2.745

QMEAN4 global scores:

QMEANscore4 Estimated absolute model quality Score components
0.604 1toh QMEAN plots Batch.1.short.pdb plot.png 1toh QMEAN plots Batch.1.short.pdb plot.png density plot.png 1toh QMEAN plots Batch.1.short.pdb plot.png slider.png


Local scores:

Coloring by residue error Residue error plot
1toh residue error structure.jpeg 1toh QMEAN plots energy profile plots Batch.1.short.pdb local energy profile QMEANlocal.png


Global scores: QMEAN4:

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:

1toh local mode quality estimation anolea qmean gromos.png



Automated Mode: Modelling with template structure 1ltz_A (<40%)

QMEAN Z-Score: -4.282

QMEAN4 global scores:

QMEANscore4 Estimated absolute model quality Score components
0.47 1ltz QMEAN plots Batch.1.short.pdb plot.png 1 ltz QMEAN plots Batch.1.short.pdb plot.png density plot.png 1 ltz QMEAN plots Batch.1.short.pdb plot.png slider.png


Local scores:

Coloring by residue error Residue error plot
1ltz residue error pdb plot.jpeg 1ltz QMEAN plots energy profile plots Batch.1.short.pdb local energy profile QMEANlocal.png


Global scores: QMEAN4:

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:

1ltz local model quality estimation anolea qmean gromos.png


Alignment Mode: Modeling with adjusted alignment 1phz_A (>60%)

QMEAN Z-Score: -2.786

QMEAN4 global scores:

QMEANscore4 Estimated absolute model quality Score components
0.6 QMEAN plots 1phz imp template.pdb plot.png QMEAN plots 1phz imp template pdb plot.png density plot.png QMEAN plots 1phz imp template plot.png slider.png


Local scores:

Coloring by residue error Residue error plot
1phz imp template coloring by residue error.jpeg QMEAN plots energy profile plots 1phz imp template.pdb local energy profile QMEANlocal.png


Global scores: QMEAN4:

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:

Local quality estimation 1phz imp template annolea qmean gromos.png


Alignment Mode: Modeling with adjusted alignment 1toh_A (>40%)

QMEAN Z-Score: -4.498

QMEAN4 global scores:

QMEANscore4 Estimated absolute model quality Score components
0.494 300px 300px 300px


Local scores:

Coloring by residue error Residue error plot
300px 300px


Global scores: QMEAN4:

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:

300px



Alignment Mode: Modeling with adjusted alignment 1ltz_A (<40%)

QMEAN Z-Score: -4.673

QMEAN4 global scores:

QMEANscore4 Estimated absolute model quality Score components
0.447 300px 300px 300px


Local scores:

Coloring by residue error Residue error plot
300px 300px


Global scores: QMEAN4:

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:

300px

iTasser

Comparison to experimental structure

To calculate the C-alpha RMSD we used DaliLite. Later we changed to the command line tool sap file1.pdb file2.pdb.

To calculate the TM-Score we used the TM-score webservice from the University of Michigan alternative. Later we changed to the command line tool TMS model.pdb native.pdb.

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 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 (sourcecode).

Modeller

Standard Workflow
Template Structure Compared To Apo/Complexed C-alpha RMSD TM score All Atoms RMSD, 6A
1PHZ Chain: A 1J8T Apo 0.9 0.9711 4.2082
1PHZ Chain: A 1J8U Complexed 0.9 0.9718 -
1TOH Chain: A 1J8T Apo 1.9 0.8844 5.9302
1TOH Chain: A 1J8U Complexed 1.9 0.8850 4.3264
1LTZ Chain: A 1J8T Apo 2.3 0.6986 1.0011
1LTZ Chain: A 1J8U Complexed 2.3 0.6985 -
Multiple 1J8T Apo 2.1 0.1886 -
Multiple 1J8U Complexed 2.1 0.1879 -
Adjusted Alignment Workflow
Template Structure Compared To Apo/Complexed C-alpha RMSD TM score All Atoms RMSD, 6A
1PHZ Chain: A 1J8T Apo 1.0 0.1957 -
1PHZ Chain: A 1J8U Complexed 1.0 0.1954 -
1TOH Chain: A 1J8T Apo 1,1 0.1592 -
1TOH Chain: A 1J8U Complexed 1.1 0.1592 -
1LTZ Chain: A 1J8T Apo 1.7 0.1021 -
1LTZ Chain: A 1J8U Complexed 1.7 0.1020 -

Swissmodel

Standard Workflow
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
Template Structure Compared To Apo/Complexed C-alpha RMSD TM score All Atoms RMSD, 6A
1PHZ Chain: A 1J8T Apo
1PHZ Chain: A 1J8U Complexed
1TOH Chain: A 1J8T Apo
1TOH Chain: A 1J8U Complexed
1LTZ Chain: A 1J8T Apo
1LTZ Chain: A 1J8U Complexed

iTasser

Standard Workflow
Template Structure Compared To Apo/Complexed C-alpha RMSD TM score All Atoms RMSD, 6A
1PHZ Chain: A 1J8T Apo
1PHZ Chain: A 1J8U Complexed
1TOH Chain: A 1J8T Apo
1TOH Chain: A 1J8U Complexed
1LTZ Chain: A 1J8T Apo
1LTZ Chain: A 1J8U Complexed


Adjusted Alignment Workflow
Template Structure Compared To Apo/Complexed C-alpha RMSD TM score All Atoms RMSD, 6A
1PHZ Chain: A 1J8T Apo
1PHZ Chain: A 1J8U Complexed
1TOH Chain: A 1J8T Apo
1TOH Chain: A 1J8U Complexed
1LTZ Chain: A 1J8T Apo
1LTZ Chain: A 1J8U Complexed

Discussion

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