Structural biology should be computable! Protein structures determined by amino acid sequences Protein structures and complexes correspond to global free energy minima Fundamental test of understanding and huge practical relevance

Model of energetics of inter and intramolecular interactions Design (Given Structure, Optimize Sequence) Prediction (Given Sequence, Optimize Structure) Ab initio structure Protein Structure Protein design prediction Protein-protein docking Protein-protein Interface design interactions ROSETTA

Model of macromolecular interactions Removal of single methyl groups can destabilize proteins --> jigsaw puzzle-like packing crucial Buried polar atoms almost always hydrogen bonded --> treat hydrogen bonding as accurately as possible Exposed charge substitutions generally have little effect --> damp long range elctrostatics Focus on short range interactions!

Random Start Low-Resolution Monte Carlo Search (integrate out sidechain degrees of freedom) High-Resolution Refinement with full atomic detail 10 5 Predictions Conformational sampling Select lowest energy models Jeff Gray (Hopkins), Ora Furman (Hebrew University), Chu Wang

Docking Low-Resolution Search Monte Carlo Search Rigid body translations and rotations Residue-scale interaction potentials Protein representation: backbone atoms + average centroids

Docking Protocol (Target 12: cohesin-dockerin; unbound-bound) 1.Initial Search2.Refinement RMSD to arbitrary starting structure Energy RMSD to starting structure of refinement (Å)

red,orange– xray blue – model; green – unbound  0.46Å interface rmsd  87% native contacts  6% wrong contacts Target 12 Cohesin-Dockerin Side Chain Flexibility dockerin cohesin Ora Furman, Chu Wang

Details of T12 Interface D39 N37 S45 L83 E86 Y74 L22 R53 dockerin cohesin red,orange– xray blue - model

red,orange– xray blue - model  0.23Å interface rmsd Target 15 immunity protein D- colicin D tRNase Accurate Side Chain Modeling colicin immunity protein Science 310,

Details of T15 Interface H611 red,orange– xray blue - model E56 K610 K608 K607 E68 E59 D61 colicin immunity protein

red,orange– xray blue – model; green – unbound  2.34Å interface rmsd  36% native contacts Target 20 HemK-RF1 Modeling Backbone Movement RF1 HemK Loop with methylated Gln Chu Wang

CASP6 T0198: PhoU domain repeat Model 2: 4A over 210 rsds (Model 1: 3.94 over 198) Phil Bradley

CASP6 T0212 Model 2: 3.97 over 109 rsds (Model 1: 4.0 over 104)

T0281 ab initio prediction (1.59Å) Phil Bradley

1r69

1ubq Science 309,

2REB

Boinc.bakerlab.org/rosetta David Kim High resolution ab initio structure prediction from single sequences by enhanced diversity “barcode” directed sampling Outreach!

High Resolution Refinement of CASP target remote homology model Calculations performed on SDSC teragrid clusters Bin Qian

High Resolution NMR Model Refinement Vatson Raman Disulfide Bond Formation Protein Blue - X-ray structure Green - NMR models Red - Rosetta models

Computing Structural Biology Free energy function reasonable => Computing simple protein structures and interactions now appears to be within reach Implications for structural genomics? More cpu power => more accurate predictions for larger proteins For larger complexes, experimental data essential (low resolution electron density!). Symmetry helps! Modeling accuracy also illustrated by structures of designed proteins

Top7 X-ray structure has correct topology. Backbone RMSD to design only 1.2Å!! C-  Backbone Overlay Red : X-ray structure Blue : Design model Brian Kuhlman, Gautam Dantas; Science

Design of novel H bond network interface G177 Q51 Q180 Q169 Y35 G177 Q180 Q169 Q51 Y35 G177 Y35 Design X-ray Lukasz Joachimiak

Design of new protein functions Design of new protein-protein interactions Design of enzymes catalyzing novel chemical reactions Design of new transcription factor and endonuclease specificities Design of HIV vaccine

HIV vaccine design Present HIV coat protein epitopes locked into conformation observed in complexes with neutralizing antibodies using designed scaffolds Preliminary results: designed proteins fold and bind neutralizing antibodies (5nM affinity). One design confirmed crystallographically. Bill Schief in collaboration with Peter Kwong

Crystal structure of Mab 2F5 in complex with its HIV epitope Model of non-HIV scaffold-epitope (red) Computational design of non-HIV immunogens to elicit broadly-neutralizing antibodies Bill Schief

WT-WT Design-WT WT-Design Design-Design Redesign of DNA cleavage specificity of MsoI homing endonuclease using ROSETTA Justin Ashworth, Jim Havranek Nature in press

Specific DNA cleavage by designed nuclease wild-type I-Mso Design - 1/2 n 1 wild-type design wild-type design Cleavage targets ½¼ - 1/2 9 5uM nuclease

Acknowledgements Design Brian Kuhlman (UNC) Gautam Dantas Justin Ashworth Jim Havranek Robetta.bakerlab.org prediction and design server: David Kim (domain parsing, boinc) and Dylan Chivian Rosetta software freely available for academic use Boinc.bakerlab.org/rosetta Protein structure prediction Phil Bradley (MIT) Rhiju Das Lars Marlstrom Bin Qian Vatson Raman Protein-protein docking Ora Furman (Hebrew University) Chu Wang Jeff Gray (Johns Hopkins)