Homology Modelling in 15 Minutes

Upload/feed your sequence into BLAST at Uniport Website>>Select template (based on

similarity and identity though sometimes people choose to select template based on active

site similarity>>Multiple sequence alignment using ClustalW (online version)>>Homology

model using Chimera>>Psipred to predict 3D secondary structure of the query sequence and

check it manually against the generated model>>Check the Ramachandran plot.

Often the generated homology model is subjected to molecular dynamics simulation in order

to minimize the clashes and to generate proper folded conformation of the protein.

Tricks:

1. Modeller the main executable software which governs the modelling stage can be

installed freely (search in GOOGLE). It will not show up in your installed

programmes list, it is just a Chimera add on whose path you need to specify during

the modelling.

Get Set and Go

1. Go to GOOGLE and type “Uniport” and click on the top result.

2. If you already have a FASTA sequence ready then paste and click on BLAST. If you

don’t know the sequence then use search the necessary. Example: Say for example

search Human Hsp90 and you will get the results something like the following image.

This search will produce the following query

3. Now choose P08238 as your query sequence and download it. Make sure you read

previous research papers to know the biological aspects.

4. After downloading the FASTA sequence or in case you already have it then click on

BLAST tab on Unipot, paste the FASTA sequence and BLAST as shown in the

following image.

It will show the job status RUNNING something like the following image

5. As soon as it is finish you will get the results something like below

6. You can see from result that a bunch of information is provided. Say for example I am

happy with P08238 (you can choose a better sequence as a template based on

sequence similarity but this choice is just for example).

7. Download the template crystal structure from RCSB PDB. In this case I choose yeast

Hsp90 PDB: 2CG9 as a template.

8. You can also find list of PDBS by the following way:

i. GOOGLE “NCBI” and click on the first result from the search. Then click on

the “Protein” tab from the drop down list and type in your search. Your search

could be either be the name of the crystal structure, FASTA code, PDB code.

Suppose I type P08238 which is a FASTA code of the homolog.

After pasting the FASTA code mentioned above you search and will get the

desired result as shown below. Click on Run BLAST as shown below

It will redirect to a page where you can run BLAST. Before running change

the preferred database to Protein Data Bank Proteins in order to get the PDB

codes and then click on BLAST.

ii. The search will produce the following results

The results shows not only the % identity and similarity but also PDB codes. This makes it easier to know more information on a particular template from PDB. Suppose I choose 2CG9, 3NMQ, 3PY and 3HJC respectively.

You can download the FASTA sequence of these PDBs from RCSB PDB or right click on the NCBI page to get it.

9. Open all the FASTA sequence in Wordpad (WINDOWS) or Textedit (Mac) and copy paste them in a text file without leaving any space as shown below

10. GOOGLE out Clustalw and click the result which is showing “Multiple sequence alignment with ClustaW). See below

11. Copy the sequences from Wordpad or Textedit and performmultiple sequence alignment. Check out the following image

12. You will get the following result if you follow properly.

13. Check the templates. Save the result by right clicking on the clusta.aln tab. You might need the picture for publication.

14. Go to Chimera>> Open the template crystal structures>>perform alignment by clicking Tools>Structure comparison>>Match maker. Choose a reference structure and click ok as shown below.

15. After match making you need to align. Go to Tools>>Structure comparison>>Match>>Align as shown below.

16. After alignment click Favourites>>Sequence>>On the new window, click Edit>> Add structure (add all templates and the query sequence).

17. To create a model from the sequence window>>Structure>>modeller>>close the query sequence>>choose templates/s>>click fetch structures/annotations (This is important because if the information concerning your templates cannot be downloaded, that means there is something wrong with your templates).

18. Click Advanced options: Run modeller locally; Number of output models: 1-5 (number depends on you)

19. Click Ok and you will see progress of your work at the bottom of Chimera.

20. Choose your model based on zDope score. Negative value indicates a better model. Choose the model with best zDope score.

21. Save the PDB generated as .pdb file. If you want to see Ramachandran plot. Download the academic version of Maestro. File>>Import structures>>Tools>>Assign bond orders>>Tools>>Ramachandran plot.

It is better to do a minimum 50ns MD simulation of the modelled structure in order to get a folded and fully minimized version of your homology model. Though research is still going strong on the applicability of homology modelling and molecular dynamics simulation.