Practical Guide to Homology Modeling: Difference between revisions

<table style="background:#ffff80;"><tr><td>Many assertions in this article are lacking literature citations. Help improving documentation in this article will be appreciated. Wikipedia's article on [http://en.wikipedia.org/wiki/Homology_modeling Homology modeling] is well documented, although more technical and less of a practical guide than the present article.</td></tr></table>

*'''&quot;Structure&quot;''' will be used in this article to mean three-dimensional structure.

You don’t need a homology model if the amino acid sequence of interest (the query sequence) already has an empirically determined 3D structure. Structures determined empirically, by X-ray crystallography or (much less often) by solution NMR, will almost always be more accurate than a homology model.

All published, empirically-determined, atomic-resolution, macromolecular 3D structures are available in the [[Protein Data Bank]] (PDB, pdb.org).

Here are two methods for finding out if your query amino acid sequence, or parts of it, have empirically-determined 3D structures in the PDB.

At UniProt.Org, find your protein and click on '''Structure''' (blue button at the left).

*If there is a section ''3D Structure Databases'' with a column labeled '''Entry''' containing 4-character PDB IDs, these are empirical structures for your protein. Pay attention to the “Positions” column, which gives the sequence number range covered by each model.

*If there is no “Entry” column, then there are no sequence-identical empirical structures for your protein. Then try the Advanced search method below.

*Some proteins have no Structure section (e.g. K4QDG1_SACBA). Then try the Advanced search method below.

#At [http://pdb.org pdb.org], go to Advanced Search.

#Click on “Choose a query type” and select Sequence under “Sequence Features”.

#Paste your query sequence into the large box, and click the “Submit Query” button at the lower right of the search interface box.

#The best hits will be listed first, starting below “Showing 1-25 of XXX Results”.  Notice that each hit starts with a large, bold PDB ID. In the “Alignment” section of the first hit, click on “Display for All Results”. Also in the “Compound” section, click “Display for All Results”.

For each hit, notice the “Identities” above the sequence alignment box. The denominator tells you the length of the sequence alignment. The percentage tells you the sequence identity of the alignment.

For example, “Identities: 355/1045 (34%)” means that 1,045 residues of your query sequence align to the hit with 34% sequence identity (355 identical residues in the alignment). Knowing that my query had length 1,170 residues, I can see that this potential template for a homology model would enable me to model 1,045/1,170 = 89% of my query sequence. Quite often the alignment would span a much smaller portion of the full-length sequence.

<font color='red'>'''BEWARE!'''</font> The sequence identity percentage may be '''underestimated''' at pdb.org. This happens when pdb.org deems segments of the query sequence to be of low complexity. Such segments are marked with X’s in the sequence alignment, and excluded from the calculation of sequence identity. For example, for Saccharomyces gal4 (UniProt P04386), for the top hit (3coq), pdb.org reports “Identities: 71/89 (80%)”, while in fact the sequence identity is 100%. Note this in the sequence alignment at pdb.org:

The 18 residues marked X were not included in the identity calculation. In contrast, when the same sequence search is performed at [http://www.ebi.ac.uk/pdbe PDB-Europe], 100% sequence identity is reported. However, other aspects of the report at PDB-Europe are less satisfactory (e.g. the length of the alignment is not stated; the sequences are not numbered) and hence we recommend using pdb.org despite its misleading sequence identity percentages. But you may certainly want to run the sequence search at [http://www.ebi.ac.uk/pdbe PDB-Europe] to compare the reported identity percentages.

Attempts to determine structure for intrinsically disordered protein will be futile. Therefore, before considering homology modeling or crystallization experiments, it is important to predict whether portions of the query protein are likely to be intrinsically disordered.

By some estimates, 10% of proteins are intrinsically disordered for their full lengths, and about 40% of eukaryotic proteins have at least one loop 50 residues or longer that is intrinsically disordered. These disordered loops are typically missing from X-ray crystallographic structures because the disorder blurs that portion of the electron density map.

==== MobiDB via UniProt ====

At UniProt.Org, find your protein, then click on “Structure”. At the bottom of this section is usually a link to MobiDB’s report for the query protein. There, in the section ''Detailed Disorder Annotations'' are graphics showing experimental evidence for disorder (if available) and, under the heading ''Predictors'', results from several servers designed to predict intrinsic disorder.

The ''Examples'' above are linked to MobiDB.

The [http://bip.weizmann.ac.il/fldbin/findex FoldIndex server] is a useful adjunct to the MobiDB report, since it is not included in that report.

Even when the sequence alignment and template result in a correct backbone fold for the homology model, the sidechain rotamer positions will be incorrect. Despite knowing where each alpha carbon atom is located, theory does not correctly predict how the sidechains will fit together. At best, the sidechain rotamer positions will avoid steric clashes and electrostatic repulsions of like charges, and may optimize some salt bridges and hydrogen bonds. However, when a high quality empirical model becomes available, the details of sidechain packing in the homology model will be shown to be incorrect.

===Pre-Calculated Models===

Under the subheading ''3D Structure Databases'', click on the linked UniProt ID at ProteinModelPortal. Here you will find bar graphics showing the coverage by pre-calculated homology models. Touching the blue bars reports the sequence range for each model.

 

 

Notice the section at the bottom of the page ''Remodel this protein''. This is a good option if you don't find a satisfactory model.

This give you similar coverage graphics, but limited to models generated by Swiss Model. Clicking on any one blue graphic bar shows details below, including links to download the model.

*Go to [http://swissmodel.expasy.org SwissModel.expasy.org].

*Download your homology model(s).

* Go to [http://firstglance.jmol.org FirstGlance.Jmol.Org].

* Click on ''Upload your own PDB file'' and designate your homology model. Click ''View in FirstGlance''. Your molecule should appear momentarily.

*Click ''Choose File'' to upload your homology model. Click ''Next''.

*Select chain A.

*Change ''number of iterations'' to 1.

*Check 'Let me select the sequences'. Leave all other settings at their defaults.

*When the sequences are gathered, you will see <font color="green">'''SELECT SEQUENCES'''</font>.