Task 4: Homology based structure predictions

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Revision as of 18:10, 3 June 2011 by Pfeiffenberger (talk | contribs) (→‎Standard Workflow)

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



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

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.

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"
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

Discussion

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