Modeling the Structures of Proteins and Macromolecular Assemblies Depts. Of Biopharmaceutical Sciences and Pharmaceutical Chemistry California Institute for Quantitative Biomedical Research University of California at San Francisco Andrej Š ali 3/25/03

1. 1.Yeast and E. coli ribosomes: Electron microscopy, comparative modeling, and structural genomics Yeast Nuclear Pore Complex: Low-resolution modeling of large assemblies; bridging the gaps between structural biology, proteomics, and system biology Comments. 4/6/03

S. cerevisiae ribosome C. Spahn, R. Beckmann, N. Eswar, P. Penczek, A. Sali, G. Blobel, J. Frank. Cell 107, , Fitting of comparative models into 15Å cryo- electron density map. 43 proteins could be modeled on 20-56% seq.id. to a known structure. The modeled fraction of the proteins ranges from 34-99%. 3/25/03

E. coli ribosome H.Gao, J.Sengupta, M.Valle, A. Korostelev, N.Eswar, S.Stagg, P.Van Roey, R.Agrawal, S.Harvey, A.Sali, M.Chapman, and J.Frank. Cell, in press. Upon EF-G binding, the ribosome becomes less compact. In contrast to mRNA, many protein contacts undergo large conformational changes, suggesting ribosomal proteins facilitate the dynamics of translation. 4/6/03

START Get profile for sequence (NR) Scan sequence profile against representative PDB chains Scan PDB chain profiles against sequence PSI-BLAST M OD P IPE : Large-Scale Comparative Protein Structure Modeling Select templates using permissive E-value cutoff 1 Expand match to cover complete domains 1 Build model for target segment by satisfaction of spatial restraints Evaluate model Align matched parts of sequence and structure MODELLER R. Sánchez & A. Šali, Proc. Natl. Acad. Sci. USA 95, 13597, N. Eswar, M. Marti-Renom, M.S. Madhusudhan, B. John, A. Fiser, R. Sánchez, F. Melo, N. Mirkovic, A. Šali. For each target sequence For each template structure 3/25/03 END

Pieper et al., Nucl. Acids Res /25/03

Comparative modeling of the TrEMBL database Unique sequences processed: 733,239 Sequences with fold assignments or models: 415,937 (57%) 4/03/02 ~4 weeks on 500 Pentium III CPUs 70% of models based on <30% sequence identity to template. On average, only a domain per protein is modeled (an “average” protein has 2.5 domains of 175 aa). 3/25/03

Model Accuracy Marti-Renom et al. Annu.Rev.Biophys.Biomol.Struct. 29, , MEDIUM ACCURACY LOW ACCURACYHIGH ACCURACY NM23 Seq id 77% CRABP Seq id 41% EDN Seq id 33% X-RAY/ MODEL Sidechains Core backbone Loops C  equiv 147/148 RMSD 0.41Å Sidechains Core backbone Loops Alignment C  equiv 122/137 RMSD 1.34Å Sidechains Core backbone Loops Alignment Fold assignment C  equiv 90/134 RMSD 1.17Å 4/6/03

Future directions Make sure we have building blocks (structural genomics). Develop methods for simultaneous fitting of proteins into EM density and conformational modeling (induced fit, comparative modeling, ab initio). Need large computing (eg, cluster of hundreds of nodes with >1GB memories). 4/6/03

Rockefeller University, New York *UCSF F. Alber*, T. Suprapto, J. Kipper, W. Zhang, L. Veenhoff, S. Dokudovskaya, M. Rout, B. Chait, A. Šali* Modeling of the yeast nuclear pore complex by satisfaction of spatial restraints (MODELLER) 4/6/03

Modeling macromolecular assemblies by satisfaction of spatial restraints 1)Representation of a system. 2)Scoring function (spatial restraints). 3)Optimization. Sali, Ernst, Glaeser, Baumeister. From words to literature in structural proteomics. Nature 422, , /25/03 There is nothing but points and restraints on them.

Modeling of NPC Stochiometry. Parse proteins into domains. Protein and “sub-complex’ shapes from Stokes radii. Excluded volume of proteins. Symmetry of NPC (EM). Radial and axial localization of proteins (IEM). Protein-protein proximity (immuno-purification). Binary protein-protein contacts from “overlay” experiments. Modeling in the context of the nuclear envelope. Comparative models for some domains. Structural genomics of nucleoporins. 4/6/03

spokehalf-spoke cytosolic side nuclear side Schematic structure of yeast NPC nuclear membrane TOP VIEWSIDE VIEW half-spoke contains ~30 nucleoporin proteins (NUPs). ~480 NUPs in NPC. C.W. Akey, M. Rout 3/25/03

[sum of residues] complex dimension f(res) [A] Protein-protein “contacts” For each protein pair within a half-spoke, the upper bound on the center-center distance is the estimated maximal complex diameter: Complications: - multiple protein copies in half-spoke - inter-spoke interactions. 3/25/03

Successful optimization 11/18/023/25/03

Scan of figure NUP120 NUP84 NUP85 NUP145C SEH1 Cdc31 Experiment Rappsilber J, Siniossoglou S, Hurt EC, Mann M. Anal Chem 2000 Jan 15;72(2): Model NUP84-complex 4/6/03

Structural proteomics aims to characterize structures of most macromolecular complexes, in space and time. On average, a domain may interact with a few other domains. The function of a complex is determined by its structure and dynamics. There are too many complexes to be determined directly by high- resolution experimental structure determination. Thus, just like in structural genomics, an efficient combination of experiment and computation is required. 4/6/03

Structural Proteomics versus Structural Genomics Potential targets not clear clear Target selectionnot clearclear Scopenot clearclear Structure determinationhybridX-ray or NMR Functional Annotationessentialnot a major focus There are additional sciences and technologies in structural proteomics, relative to structural genomics. 4/6/03

Structural Genomics Characterize most protein sequences based on related known structures. There are ~16,000 30% seq id families (90%) (Vitkup et al. Nat. Struct. Biol. 8, 559, 2001) Sali. Nat. Struct. Biol. 5, 1029, Sali et al. Nat. Struct. Biol., 7, 986, Sali. Nat. Struct. Biol. 7, 484, Baker & Sali. Science 294, 93, The number of “families” is much smaller than the number of proteins. Any one of the members of a family is fine. Characterize most protein sequences based on related known structures: 3/25/03

Target selection for structural proteomics (scope) Comprehensive coverage. What targets: Stable assemblies? Transient complexes? Pairs of domains? Can they be organized into groups? How many such groups are there? 4/6/03

Target selection for structural proteomics? There are ~3,000 folds containing ~90% of all sequences. A target: Binary domain-domain interface. There may be a finite number of domain-domain interfaces, largely defined by the domain fold types. Given pairwise interactions, a large assembly may be reconstructed. 4/6/03

Protein and assembly structure by experiment and computation 3/25/3 Sali, Ernst, Glaeser, Baumeister. From words to literature in structural proteomics. Nature 422, , /25/03

Modeling macromolecular assemblies by satisfaction of spatial restraints 1)Representation of a system. 2)Scoring function (spatial restraints). 3)Optimization. Sali, Ernst, Glaeser, Baumeister. From words to literature in structural proteomics. Nature 422, , /25/03 There is nothing but points and restraints on them.

Future directions Development of general/flexible/hierarchical representation of assemblies. Quantifying spatial information from experiments. Optimization of structure. Toy models. Space, time. 4/6/03

Role of NIH Structural proteomics is timely and feasible. Humongous integration of concepts, sciences, methods, tools, people. Thus, need research centers, but also “R01” research. Significant computing. Bridging the gaps between structural biology, proteomics, and system biology. 4/6/03