Homology Modelling GLA

^{by Benjamin Drexler and Fabian Grandke}

Calculation of Models

Available Homologous Structures

The following table lists the best ten hits of the HHpred search of Task 1. We used 3HG3 (97% identity), 1KTB (53%) and 3CC1 (34%) as templates for the homology modelling.

PDB-ID	Name	Probability	E-value	P-value	Identity	Template
> 60% sequence identity
3hg3_A	Alpha-galactosidase A	1.0	0	0	97%	x
> 40% sequence identity
1ktb_A	Alpha-N-acetylgalactosaminidase	1.0	0	0	53%	x
< 40% sequence identity
1uas_A	Alpha-galactosidase	1.0	0	0	39%
3lrk_A	Alpha-galactosidase 1	1.0	0	0	32%
3a5v_A	Alpha-galactosidase	1.0	0	0	35%
1szn_A	Alpha-galactosidase	1.0	0	0	34%
3a21_A	Putative secreted alpha-galactosidase	1.0	0	0	34%
3cc1_A	BH1870 protein	1.0	0	0	26%	x
3a24_A	Alpha-galactosidase	1.0	0	0	14%
1zy9_A	Alpha-galactosidase	1.0	2.2E-37	8.8E-42	14%

MODELLER

We used MODELLER as described in the tutorial Using Modeller for TASK 4.

Pairwise Alignments

In this section, we used a pairwise alignment between the template (i.e. 3HG3, 1KTB and 3CC1) and the target as the input for MODELLER.

Evaluation

	Apo (1R46)			Complexed (1R47)
Template	TMS	RMSD	RMSD catalytic site	TMS	RMSD	RMSD catalytic site
3HG3	0.141	0.498	0.326	0.1413	0.512	0.366
1KTB	0.140	0.901	0.439	0.1413	0.888	0.437
3CC1	0.1397	2.864	3.436	0.1397	2.853	3.405

Representation of the resulting models of MODELLER and the reference PDB structure 1R47. The models are in superposition to the reference structure (green) and are shown in cartoon representation. (A) The model is based on the PDB structure 3HG3 (red). (B) The model is based on the PDB structure 1KTB (blue). (C) The model is based on the PDB structure 3CC1 (magenta).

Multiple Sequence Alignments

PDB-ID	Unsupervised	Supervised	Identity	Comment
3LX9_A			99%
3GXP_A			99%
3H53_A			99%
3HG3_A			97%
3IGU_A			54%
1KTB_A			53%
1UAS_A			39%
3LRK_A			34%	Was removed due to little sequence identity. Caused huge gaps in alignment.
3CC1_A			28%	Was removed due to little sequence identity. Caused huge gaps in alignment.

iTasser

Figure 1 shows, that iTasser takes an amino acid sequence as input and tries to retrieve template proteins from PDB. In the next step fragments from the the templates are reassembled to a complete model. In the last step, the model is reassembled by taking energy calculations into account. Additionally biological function prediction is done, but that was not of interest of this task.<ref name=itasser1>http://zhanglab.ccmb.med.umich.edu/I-TASSER/about.html</ref>

Figure 1: A schematic representation of the I-TASSER protocol for protein structure and function predictions. The protein chains are colored from blue at the N-terminus to red at the C-terminus.<ref name=itasser2>Roy et al., I-TASSER: a unified platform for automated protein structure and function prediction, Nature Protocols, 2007</ref>

We used the iTasser-server in two different ways:

1. Standard parameters: the protein sequence is given as input and the program searches PDB for templates. The found proteins are used to create a template to predict the structure.
2. PDB-ID as input: together with the amino acid sequence a template PDB-ID is given as input. The program takes all available information into account and uses them to calculate the structure.

As the iTasser server has very low capacities and only one job commitment at the same time is possible, the results of the second way are not yet present. The standalone version is no option, because it has a size of about 10GB and it does not work properly.

SWISS-MODEL

We used the swissmodel server with two different options:

1. Automated Mode: A template sequence is given as input. As no further information are given, the model is directly created from the amino acid sequence. This method should only be used, if the sequence identity between target and template is greater than 50%.
2. Aligned Mode: A pairwise alignment of template and target sequence is given as input. We created our alignments using online ClustalW2 from EBI.

Following sequences have been selected:

3hg3_A					1ktb_A					3cc1_A
	Automated Mode		Aligned Mode			Automated Mode		Aligned Mode			Automated Mode		Aligned Mode
Identity	Z-score	Model	Z-score	Model	Identity	Z-score	Model	Z-score	Model	Identity	Z-score	Model	Z-score	Model
97%	0		-0.415		53%	0		-2.261		26%	0		Error¹	NA

¹The sequences are to different to create a useful model from the alignment. In the automated mode the template itself has been used as model, what is useless, because the sequences have only 26% identity.

Aborting: too many unfruitful attempts to rebuild a loop. This is likely to indicate a misalignment in this region Use Swiss-PdbViewer to adjust your alignment in this region and resubmit an optimise mode modelling request.

Evaluation of Models

MODELLER

Numeric Evaluation

	Apo (1R46)			Complexed (1R47)
Template	TMS	RMSD	RMSD catalytic site	TMS	RMSD	RMSD catalytic site
3HG3	0.141	0.498	0.326	0.1413	0.512	0.366
1KTB	0.140	0.901	0.439	0.1413	0.888	0.437
3CC1	0.1397	2.864	3.436	0.1397	2.853	3.405

Comparison to Experimental Structure

Representation of the resulting models of MODELLER and the reference PDB structure 1R47. The models are in superposition to the reference structure (green) and are shown in cartoon representation. (A) The model is based on the PDB structure 3HG3 (red). (B) The model is based on the PDB structure 1KTB (blue). (C) The model is based on the PDB structure 3CC1 (magenta).

SWISS-MODEL

Template 3HG3

Numeric Evaluation

	Apo (1R46)			Complexed (1R47)
Mode	TMS	RMSD	RMSD catalytic site	TMS	RMSD	RMSD catalytic site
Aligned	0.1411	0.485	0.279	0.1412	0.489	0.290
Automated	0.1411	0.485	0.277	0.1412	0.489	0.291

Comparison to Experimental Structure

Representation of the resulting models of SWISS-MODEL which used the PDB structure 3HG3 as a template the reference PDB structure 1R47. The models are in superposition to the reference structure (green) and are shown in cartoon representation. (A) The model (cyan) was build by using the aligned mode of SWISS-MODEL. (B) The model (yellow) was build by using the aligned mode of SWISS-MODEL.

Template 1KTB

Numeric Evaluation

	Apo (1R46)			Complexed (1R47)
Mode	TMS	RMSD	RMSD catalytic site	TMS	RMSD	RMSD catalytic site
Aligned	0.1598	0.943	5.073	0.1606	0.932	6.409
Automated	0.1361	0.981	0.417	0.1368	0.974	0.404

Comparison to Experimental Structure

Representation of the resulting models of SWISS-MODEL which used the PDB structure 1KTB as a template and the reference PDB structure 1R47. The models are in superposition to the reference structure (green) and are shown in cartoon representation. (A) The model (red) was build by using the aligned mode of SWISS-MODEL. (B) The model (blue) was build by using the aligned mode of SWISS-MODEL.

Template 3CC1

Numeric Evaluation

	Apo (1R46)			Complexed (1R47)
Mode	TMS	RMSD	RMSD catalytic site	TMS	RMSD	RMSD catalytic site
Aligned	0.1302	3.279	7.107	0.1300	3.802	7.357
Automated	N/A	N/A	N/A	N/A	N/A	N/A

Comparison to Experimental Structure

Representation of the resulting model of SWISS-MODEL which used the PDB structure 3CC1 as a template and the reference PDB structure 1R47. The model is in superposition to the reference structure (green) and are shown in cartoon representation. It was not possible to build a model with the automated mode of SWISS-MODEL and hence there is only a model of the aligned mode (magenta).

References

<references />