Task 4: Homology based structure predictions

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Revision as of 14:15, 3 June 2011 by Pfeiffenberger (talk | contribs) (Swissmodel)

Task description

The full description of this task can be found here.


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

Homology modelling with Modeller

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

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

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

iTasser

Comparison to experimental structure

Modeller

Swissmodel

To calculate the C-alpha RMSD we used DaliLite.

To calculate the TM-Score we used the TM-score webservice from the University of Michigan.

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
1PHZ Chain: A 1J8U Complexed 0.9 0.7408
1TOH Chain: A 1J8T Apo 1.3 0.8889
1TOH Chain: A 1J8U Complexed 1.2 0.8894
1LTZ Chain: A 1J8T Apo 2.3 0.8816
1LTZ Chain: A 1J8U Complexed 2.3 0.8814
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

Discussion