Task 4: Homology based structure predictions

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 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)
> 40% sequence identity	1TOH Chain: A	P00439 (Phenylalanine-4-hydroxylase)
< 40% sequence identity	1LTZ Chain: A	P00439 (Phenylalanine-4-hydroxylase)

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

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

Local scores:

Coloring by residue error	Residue error plot

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:

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

Local scores:

Coloring by residue error	Residue error plot

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:

Comparison to experimental structure

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