Molecular modelling Practical session José R. Valverde CNB/CSIC jrvalverde@cnb.csic.es © José R. Valverde, 2014 CC-BY-NC-SA

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

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

Servers CPHmodels http://www.cbs.dtu.dk/services/CPHmodels/ HHpred http://toolkit.tuebingen.mpg.de/hhpred ModBase http://modbase.compbio.ucsf.edu/ LOOPP http://cbsuapps.tc.cornell.edu/loopp.aspx Zhang Lab http://zhanglab.ccmb.med.umich.edu/

Servers (continued) Phyre2 http://www.sbg.bio.ic.ac.uk/~phyre2/ (ps)2 http://ps2.life.nctu.edu.tw/ PsiPred http://bioinf.cs.ucl.ac.uk/psipred/ M4T http://manaslu.aecom.yu.edu/M4T/ SwissModel http://swissmodel.expasy.org/

Threading software HHpred http://toolkit.tuebingen.mpg.de/hhpred RaptorX http://raptorx.uchicago.edu/ Phyre2 http://www.sbg.bio.ic.ac.uk/phyre2 SPARKSX sparks-lab.org/yueyang/server/SPARKS-X/ 3D-JigSaw http://bmm.cancerresearchuk.org/~3djigsaw/

Ab initio modeling Some servers Quark:zhanglab.ccmb.med.umich.edu/QUARK/ Robetta: http://robetta.bakerlab.org/ Evfold: http://evfold.org/evfold-web/evfold.do

D I Y

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 http://salilab.org/modeller/

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 http://www.ncbr.muni.cz/triton/

In silico mutagenesis

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

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

Open TRITON Open a terminal and type “triton &”

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

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

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

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

Select desired mutation Click on “Ala” and press Forward

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

Run calculation Click on Run calculation automatically Press Finish

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

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”

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)

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

The End? Image from PublicDomainPictures. CC0 http://pixabay.com/en/camel-oman-egypt-morocco-wallpaper-163703/

A new beginning Image from PublicDomainPictures. CC0 http://pixabay.com/en/arizona-sunrise-light-landscape-165094/