1. Molecular modelling Practical session
José R. Valverde
CNB/CSIC
© José R. Valverde, 2014
CC-BY-NC-SA

2. A tour of servers Building models today can be very easy You need
A sequence in FASTA format (or maybe the sequence alone)
An address (to receive the results)
Patience
How-to:
Connect to modeling server
Enter your address and paste your sequence
Click on “Submit” or equivalent

3. MetaServers GeneSilico www.genesilico.pl/meta2/
Wide variety of predictions
LOMETS:
zhanglab.ccmb.med.umich.edu/LOMETS
Local MetaThreading server
Protein Model Portal:
Interactive Modeling
Access existing models

4. Servers CPHmodels http://www.cbs.dtu.dk/services/CPHmodels/ HHpred
ModBase
LOOPP
Zhang Lab

5. Servers (continued) Phyre2 http://www.sbg.bio.ic.ac.uk/~phyre2/ (ps)2
PsiPred
M4T
SwissModel

6. Threading software HHpred http://toolkit.tuebingen.mpg.de/hhpred
RaptorX
Phyre2
SPARKSX
sparks-lab.org/yueyang/server/SPARKS-X/
3D-JigSaw

7. Ab initio modeling Some servers
Quark:zhanglab.ccmb.med.umich.edu/QUARK/
Robetta:
Evfold:

8. D I Y

9. MODELLER A constrains-based molecular modeling program
Freely available (but you must register) for academic use
Requires a key to unlock (you'll get it after successful registration)
Python and shell programming highly advisable

10. TRITON A GUI to various common tasks, among them protein modeling
Uses standard stock “scripts” to perform its work
Generally useful for in silico mutagenesis
You can study and change the scripts
Free for academic use, but requires registration
Requires a license for MODELLER

11. In silico mutagenesis

12. Determine relevant a.a. Launch UCSF Chimera
Open Documents/epsilon-zeta/3Q8X.pdb
Select → Residue → UD1
Tools → Surface/Binding Analysis → FindHBond
Label H-bond with distance
Relax H-bond constrains
Color H-bonds not meeting precise criteria differently
Only find H-bonds with at least one end selected
Do not include intra-residue H-bonds
If end-point atom is hidden, show endpoint residue

14. Select a. a. to mutate Look at the active site
Select an amino acid to mutate
Note the amino acid type, number and chain
Tips:
Select → Clear selection
Actions → Ribbon → Hide
Thr 118 D has two hydrogen bonds to the substrate
Quit UCSF Chimera

16. Open TRITON
Open a terminal and type “triton &”

17. Create a new mutagenesis project
Click on New Project
Select Mutagenesis to launch the wizard

18. Choose a project name Read the explanation and press “Forward”
Choose a project name (e. g.T118A)
Place project to directory
“~/Documents/epsilon-zeta”
Press Forward

20. Select structure to mutate
Select the PDB file
~/Documents/epsilon-zeta/3Q8X.pdb
Press Forward

21. Select Residue to mutate
Look for 118 Thr D and add (→) it to the list
Press Forward

22. Select desired mutation
Click on “Ala” and press Forward

23. Set modeller parameters
For speed, we will use Mol. Dynamic level:
very fast
We assume the mutant does also bind the substrate
Read HETATM coordinates
Include/add H in the model
Press Forward

24. Run calculation
Click on Run calculation automatically
Press Finish

25. Wait for green light Click on the project to see its status
You can exit Triton while the calculation runs
Wait for the green check mark: done

26. Look under the hood Go to a terminal Move to the working directory
cd Documents/epsilon-zeta/T118A/T118A/Mutagenesis/
Look around: “ls”
Look at the alignment: “less mutant.ali”
Look at the modeller script: “less model.py”
Look at modeller log file: “less model.log”

27. Check mutation effects
Open UCSF Chimera
Open the results file
Documents/epsilon-zeta/T118A/T118A/Mutagenesis/
mutantT118A.pdb
Check the number of H-bonds with amino acid A118
Tips:
select ligand residue (UD1)
Compute H-bonds and save to Reply-Log
Check Reply Log (Favorites → Reply Log)

28. Draw your own conclusions
How many H-bonds does the mutant stablish with the ligand?
Are still there the H-bonds between T118 and UD1?
Are there more or less overall H-bonds?
Consider that Modeller treats the ligand as a rigid molecule and we used a very fast MD optimizer
The ligand may also adapt to the protein moiety
Affinity and reactivity also depend on other factors

29. The End? Image from PublicDomainPictures. CC0

30. A new beginning Image from PublicDomainPictures. CC0