Applications of Homology Applications of Homology ModelingModeling

Hanka Venselaar

This seminar….

Homology Modeling…• Why? • What?• When?• How?

• And a few real world examples….

Hearing loss

No structure:MGTPWRKRKGIAGPGLPDLSCALVLQPRAQVGTMSPAIALAFLPLVVTLLVRYRHYFRLLVRTVLLRSLRDCLSGLRIEERAFSYVLTHALPGDPGHILTTLDHWSSRCEYLSHMGPVKGQILMRLVEEKAPACVLELGTYCGYSTLLIARALPPGGRLLTVERDPRTAAVAEKLIRLAGFDEHMVELIVGSSEDVIPCLRTQYQLSRADLVLLAHRPRCYLRDLQLLEAHALLPAGATVLADHVLFPGAPRFLQYAKSCGRYRCRLHHTGLPDFPAIKDGIAQLTYAGPG

DFNB 63 Sequence:

Homology modeling in short…Prediction of structure based upon a highly similar structure

2 basic assumptions:

•Structure defines function

•During evolution structures are more conserved than sequence

Use one structure to predict another

Homology modeling

Example: by 80 residues 30% identity sufficient

# residues

% id

entit

y

*

* Actually, modelling is possible, but we cannot get an alignment…

O

Homology modeling in short…Prediction of structure based upon a highly similar structure

Add sidechains, Molecular Dynamics simulation on model

Unknown structure

NSDSECPLSHDG

NSDSECPLSHDG

|| || | ||

NSYPGCPSSYDG

Alignment of model and template sequence

Known structure

Known structure Back bone copiedCopy backbone and conserved residues

Model!

The 8 steps of Homology modeling

1: Template recognition and initial alignment

1: Template recognition and initial alignment

• BLAST your sequence against PDB

• Best hit normally template

• Initial alignment

NSDSECPLSHDGYCLHDGVC

|| || | ||||| |||

NSYPGCPSSYDGYCLNGGVC

1: Template recognition and initial alignment

2: Alignment correction

2: Alignment correction

• Functional residues conserved• Use multiple sequence alignments• Deletions shift gaps

CPISRTGASIFRCW CPISRTGASIFRCWCPISRTA---FRCW CPISRT---AFRCW

CPISRTAAS-FRCWCPISRTG-SMFRCWCPISRTA--TFRCWCPISRTAASHFRCWCPISRTGASIFRCW CPISRTA---FRCW

Both are possible

Multipe sequence alignment

Correct alignment

Sequence with known structure

Your sequence

2: Alignment correction

• Core residues conserved• Use multiple sequence alignments• Deletions in your sequence shift gaps

Known structure FDICRLPGSAEAV

Model FNVCRMP---EAI

Model FNVCR---MPEAI

S

G

P

L

A

E

R

C

I V

C

R

M

P

EV

C

R M

P

E

Correct alignment

F-D--A-V

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

3: Backbone generation

• Making the model….• Copy backbone of template to model• Make deletions as discussed• (Keep conserved residues)

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

4: Loop modeling

Known structure GVCMYIEA---LDKYACNC

Your sequence GECFMVKDLSNPSRYLCKC

Loop library,

try different options

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

5: Side-chain modeling

• Several options• Libraries of preferred rotamers based

upon backbone conformation

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

6: Model optimization

• Molecular dynamics simulation• Remove big errors

• Structure moves to lowest energy conformation

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

7: Model validation

7: Model Validation

• Second opinion by PDBreport /WHATIF• Errors in active site? new alignment/

template

• No errors? Model!

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

7: Model validation

8: Iteration8: Iteration

8: Iteration

8: Iteration

Model!

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

7: Model validation

8: Iteration8: Iteration

8: Iteration

8: Iteration

8 steps of homology modeling1: Template recognition and initial alignment2: Alignment correction3: Backbone generation4: Loop modeling5: Side-chain modeling6: Model optimization7: Model validation8: Iteration

Alignment

Modeling

Correction

Hearing loss

Structure!MGTPWRKRKGIAGPGLPDLSCALVLQPRAQVGTMSPAIALAFLPLVVTLLVRYRHYFRLLVRTVLLRSLRDCLSGLRIEERAFSYVLTHALPGDPGHILTTLDHWSSRCEYLSHMGPVKGQILMRLVEEKAPACVLELGTYCGYSTLLIARALPPGGRLLTVERDPRTAAVAEKLIRLAGFDEHMVELIVGSSEDVIPCLRTQYQLSRADLVLLAHRPRCYLRDLQLLEAHALLPAGATVLADHVLFPGAPRFLQYAKSCGRYRCRLHHTGLPDFPAIKDGIAQLTYAGPG

DFNB 63 Sequence:

Saltbridge between Arginine andGlutamic acid is lost in both cases

•Arginine 81 -> Glutamic acid

•Glutamic acid 110 -> Lysine

Mutations:

Mutation:

•Tryptophan 105 -> Arginine

Hydrophobic contacts from the Tryoptohan are lost, introduction of an hydrophilic and charged residue

The three mutated residues are all important for the correct positioning of Tyrosine 111

Tyrosine 111 is important for substrate binding

Accepted in Nature Genetics

Homology Modeling…• What? Prediction of an unknown structure based on an

homologous and known structure• Why? To answer biological and medical questions when

the “real” structure is unknown• When? A template with enough identity must be available• How? 8 Steps

• Real world examples: mutations in DFNB63 gene can lead to hearing disorders.

To conclude….

And now….• Go to the course website:

http://swift.cmbi.ru.nl/teach/graduateCourse• Follow the steps on the site• A few hints….

Login with username: c01 – c13 (number of your pc is written on the pc itself)

Click on the icon to open a terminal

Run Yasara by typing “yasara” in that terminal

Save your files on the Desktop