RosettaSteven Bitner

Objectives

Introduction How Rosetta works How to get it How to install/use it

Introduction

Developed in the David Baker lab at University of Washington Winner of CASP (Critical Assessment of Structure Prediction)

competition at Lawrence Livermore Labs, CA Implies that Rosetta is the best de novo predictor

Rosetta is a protein prediction and docking software package Also used to design proteins from (nearly) arbitrary 3-D shapes

November, 2003, ‘Top-7’ first synthetic protein Rosetta home

Human Proteome Folding Project Also called the World Community Grid

TOP-7 protein designed, or synthesized, using Rosetta

How Rosetta Works

Minimize energy in the folded state Uses a combination of energy formulas based on the likelihood

of particular structures, and the fitness of the sequence Side-chains simplified to a centroid located at center of

mass of the side-chain Average of observed side-chain centroids in known structures

Local sequence does not decide the local structure, it only biases the decision

Non-local favorable conditions Buried hydrophobic fragments Paired β strands Specific side-chain interactions

How Rosetta Works (Energy functions)

How Rosetta Works (Energy functions) cont.

How Rosetta Works cont.

Side-chains are added using Monte Carlo methods

Overlaps of side-chain centroids and backbone atoms are penalized

Uses probabilistic β-strand pairing and β-sheet patterns

Fragment Insertion - more later Fragment Assembly - more later

Fragment Insertion

Finds three and nine residue fragments from known library and replaces unknown torsion angles with the ‘known’ ones

Scores all windows of three and nine residues

Create fragment list with the 200 best three residue and 200 best nine residue fragments

Fragment Assembly

Randomly choose a nine residue fragment from the top 25 fragments in the ranked list Score this replacement, negatives are kept

Each simulation chooses a different random start and attempts 28,000 nine residue insertions

Next 8,000 attempted three residue insertions are scored with the overall structure

Why it’s Fast

Changes multiple angles simultaneously by using fragments from the library

Angular changes are discrete, not continuous

Getting the Software

Go to the website (bakerlab.org) Register by clicking on Rosetta Licensing

Information Go to link in email that is sent to you Download

Installing software

Upload onto a Linux machine, or other supported platform

(see README_platform) UTD’s Apache server does not work

Unpack using tar –zxvf ‘filename’ Go into rosetta++ directory Make gcc

Takes about 20 minutes This is the standard version

Different install versions

Other ways to install than make gccSee the README in rosetta++ directoryGCCDEBUG – for use if you plan on

making updates to the software

Using the downloaded software

PDB file must be in the same directory as the program or the paths.txt file must be updatedpaths.txt must be updated for the data source

the default is a non-existant directory User guide – assumes a good knowledge

of the system

Using Rosetta

Rosetta on-line Server – 200 residues at a time http://rosettadesign.med.unc.edu/index.html

Robetta site – down until mid October ’06http://robetta.bakerlab.org/

Downloaded software Can use res files to specify portions of the

backbone, or you can select the residues that you wish to pack on the web server

Interpreting results

Output file fieldsRosetta Commons site also has similar

document except the energy labels use E for energy in stead of LJ and LK for Lennard-Jones and Lazaridis-Karplus respectively as the prefix

E.g. Lennard-Jones attractive score is Eatr in the Rosetta commons output file and LJatr in the Rosetta Design output file

Input PDB file 1HOE– displayed using PyMol

Output PDB file 1HOE – displayed using PyMol

1 u b q

Before → After

Software used for this presentation

Rosetta – release 2.1.0 Rosetta Design Server

http://rosettadesign.med.unc.edu/index.html PyMol for visualization RCSB PDB

http://www.rcsb.org/pdb/Welcome.do

References

Rosetta Design Web Server http://rosettadesign.med.unc.edu/documentation.html

Protein Structure Prediction Using Rosetta, Numerical Computer Methods, C.A. Rohl, C.E. Strauss, K.M. Misura, D. Baker, pp. 66-93, 2004

README documentation included with rosetta2.0.1 Rosetta Website https://www.rosettacommons.org/ David Baker Lab Homepage http://www.bakerlab.org/