Homology Modeling


February 22th, 2007

Torsten Schwede (Torsten.Schwede@unibas.ch)
Lorenza Bordoli (Lorenza.Bordoli@unibas.ch)



1) Homology Modeling


The human transmembrane protease (Swiss-Prot P57727) consists of 3 domains: the LDL receptor domain, the SRCR domain and the serine protease domain. Information about the location and the function of the different domains, can be retrieved by consulting the InterPro entry for that protein.

We are going to model the LDL domain of the protein, whose amino acid sequence can be downloaded here [txt]. Please save a file with the sequence locally on your PC.

1) First of all we are going to look for a suitable template, e.g. using SAM HMM search algorithm accessible from the Tools section (Template identification) of the SWISS-MODEL Workspace. You can use the SWISS-MODEL Workspace anonmously or as a registered user. If you would like to use the server anonymously please remember to bookmark the page of the results. How many different hits do you detect? How do they differ? To build the model we will use the structure corresponding to the PDB entry 2gtl chain N as template. Please save chain N of the pdb entry 2gtl locally on your PC.

2) In the second step we are going to build an alignment between the target and the template sequence. Multiple sequence alignments between target, template and related sequences perform better than a simple target/template pair-wise sequence alignmnet. Therefore we are going to search for related sequences, e.g. by running a BLAST against the Swiss-Prot protein sequence database. Multiple sequence alignment (MSA) of the LDL domain sequences can be then calculated using the T-Coffee MSA tool. Please be carful to align only the regions of the different homologous sequences which correspond to the LDL domain.We will use the information from this MSA to adjust the alignment between our target sequence and the template (2gtlN).

3) Once we have obtained the MSA alignment we can prepare with the help of DeepView a so called project file containing the alignment between the target sequence and the template sequence. This project file will be used by SWISS-MODEL to build a 3D model for the LDL receptor of our target protein P57727.

3) To produce a DeepView project file to be submitted to the SWISS-MODEL homology modeling server please follow these steps:
- Load your target sequence (contained in a flat/text file): DeepView-> SwissModel->Load Raw Sequence to Model...
- Load the template file (2gtl_N) into DeepView (File-> Open PDB File ...)
- Fit the target to the template ( Fit->Fit Raw Sequence)
- Carefully check the alignment (DeepView->Window->Alignment) between your target and the template sequences. If needed, amend the alignment *.
- Then save a "Project" file (File->Save->Project...)
- And submit this file to the Project Mode of the SWISS-MODEL Workspace, in the Modeling section of the server.

* To amend the alignment check: Prosite Patterns location, disulfid bridges, use the information of a multiple sequence alignment of related sequences, ...

Once you have obtained the model, please answer the following questions:
Are you sure that your model has the correct protein fold? Which structural features are characteristic for this protein domain?
A protein sequence variant D(103)G has been described (D(103) of the full length protein correspond to D(34) in the LDL domain). What do you think is the function of the mutated residue in the native (not mutated) protein domain? Do you think the mutation will affect the function of this protein domain? Why? Hint: superpose the model and the template structure (DeepView-> Fit-> Iterative magic Fit) and check the residues around the mutated residue.


2) Model Evaluation

A 3D model of the Drosophila UDP-glucose 4-epimerase protein has been generated by homology modeling. The structure of the Human homolog protein Q14376 has been used as template. The PDB ID for the template is 1ek5. Two different models, Model1 and Model2 have been obtained: they differ in the alignment between the target and the template.
Evaluate the two models by checking the following criteria:

Which of the model would you trust more and why?



Visualization and analysis tool:

DeepView: [tutorial][download]

Web servers:

Databases:

PDB: repository for 3-D biological macromolecular structure data.
SWISS-MODEL Repository: Database of annotated 3D comparative protein structure models.
Swiss-Prot: Protein knowledgebase.


Output of the different web based programs (as of 22.2.07):

Template Identification [output]
Multiple Sequnce Alignment [input] [output]
SWISS-MODEL results (project mode) [input] [output html][output pdb]
ANOLEA output for Model1 [html]
ANOLEA output for Model2 [html]



Questions and comments can be sent to Lorenza.Bordoli@unibas.ch