Marc A. Marti-Renom Genome Biology Group (CNAG) Structural - PowerPoint PPT Presentation

Hybrid [integrative] methods for structure determination � � Marc A. Marti-Renom Genome Biology Group (CNAG) Structural Genomics Group (CRG)

IMP

Integrative Modeling Since 1956 Francis Crick James Watson Maurice Wilkins Rosalind Franklin

Build geometric models Spatial restraints X-ray NMR Electron Small-angle Cross-linking crystallography microscopy x-ray scattering HDXMS Proteomics, Copurification Bioinformatics, mass spectrometry physics Sampling and analysis Components Model

The Integrative Modeling Platform (IMP) http://www.integrativemodeling.org Simplicity Generalization Chimera tools/ web apps Domain-specific applications multifit/restrainer IMP C++/Python library Russel et al, PLoS Biology, 2012

The Integrative Modeling Platform (IMP) http://www.integrativemodeling.org Stage 1 : Gathering information. Stage 2 : Choosing how to represent and evaluate models. Stage 3 : Finding models that score well. Stage 4 : Analyzing resulting models and information. IMP Russel et al, PLoS Biology, 2012

Representation • Atomic • Rigid bodies • Coarse grained • Multi-scale • Symmetry/periodicity • Multi-state systems

Scoring Proteomics • Density maps • EM images • FRET • Chemical cross linking • Homology-derived restraints • SAXS • Native mass spec • Statistical potentials • Molecular mechanics forcefields • Bayesian scoring functions • Library of functional forms (ambiguity, ...) •

Sampling • Monte-Carlo • Conjugate Gradients • Quasi-Newton • Simplex • Divide and conquer sampler

Analysis • Clustering • Output • Chimera • Pymol • PDBs • Density maps

��������������������� �� The NPC Alber, F., Dokudovskaya, S., Veenhoff, L. M., Zhang, W., Kipper, J., Devos, D., Suprapto, A., et al. (2007). Nature, 450(7170), 695–701

� � � � � � � � � � � �� ��������������������� � Representation � � � 436 proteins! � } � } � � 1 N � 2 � 2 � 1 { B j n � { B j n � N � N � N � r r 1,2,5 2 3.0 1,5 9 1.5 � � Nup192 1 1 3 - 1 - 2 2 1.5 � Nup1 0 1 1,2,5 2 3.0 3 - 1 - � Nup188 1 1 3 - 1 - 4 7 1.5 � 1,2,5 2 2.9 1,5 12 1.3 � � Nup170 1 1 3 - 1 - 2 3 1.3 � Nsp1 2 2 1,2,5 3 2.5 3 - 1 - � Nup157 1 1 3 - 1 - 4 9 1.3 � 1,2,5 2 2.7 1,2,5 2 2.1 � � Nup133 1 1 Gle1 1 0 3 - 1 - 3 - 1 - 1,2,5 2 2.6 1,5 4 1.6 � � Nup120 1 1 Nup60 0 1 3 - 1 - 2,3 1 1.6 � 1,2,5 3 2.0 4 3 1.6 � � Nup85 1 1 3 - 1 - 1,5 4 1.6 � 1,2,5 3 2.0 2 2 1.6 � � Nup84 1 1 Nup59 1 1 3 - 1 - 3 - 1 - 1,2,5 2 2.3 4 2 1.6 � � Nup145C 1 1 3 - 1 - 1,5 3 1.8 � Nup57 1 1 Seh1 1 1 1,2,3,5 1 2.2 2,3 1 1.8 � � Sec13 1 1 1,2,3,5 1 2.1 4 2 1.8 � � Gle2 1 1 1,2,3,5 1 2.3 1,5 3 1.7 � � 1,2,5 2 2.4 Nup53 1 1 2,3 1 1.7 � � Nic96 2 2 3 - 1 - 4 2 1.7 � 1,2,5 2 2.3 Nup145N 0 2 1,5 6 1.5 � � Nup82 1 1 3 - 1 - 2,3 1 1.5 �

�� ��������������������� Scoring Data generation Data interpretation Method Experiments Restraint R C R O R A Functional form of activated feature restraint Bioinformatics and Membrane fractionation sequences Protein-protein : 30 nup Violated for f < f o . f is the distance between two beads, f o is the sum of the bead radii, Protein excluded volume 1,864 and �� is 0.01 nm. restraint - - * 1,863/2 Applied to all pairs of particles in representation � =1: B ms � B j � � � 1 � , s , � , i � � � sequences Membrane-surface location: 30 nup Violated if f � f o . f is the distance between a protein particle and the closest point on the - - 48 NE surface (half-torus), f o = 0 nm, and �� is 0.2 nm. Applied to particles: � � B ms � B j � � 6 � , s , � , i � � | � � (Ndc1,Pom152,Pom34) Surface localization restraint Pore-side volume location: sequences and Violated if f < f o . f is the distance between a protein particle and the closest point on the immuno-EM (see below) - - 64 NE surface (half-torus), f o = 0 nm, and �� is 0.2 nm. Applied to particles: 30 Nup B ms � B j � � � 8 � , s , � , i � � � | � � (Ndc1,Pom152,Pom34) Perinuclear volume location: Violated if f > f o ,, f is the distance between a protein particle and the closest point on - - 80 the NE surface (half-torus), f o = 0 nm, and �� is 0.2 nm. Applied to particles: B ms � B j � � 7 � , s , � , i � � � � � (Pom152) � Complex diameter Complex shape restraint 1 S-value Violated if f < f o . f is the distance between two protein particles representing the largest diameter of the largest complex, f o is the complex maximal diameter D=19.2-R , where Hydrodynamics 1 164 1 R is the sum of both particle radii, and �� is 0.01 nm. Applied to particles of proteins in experiments composite C 45 : B ms � B j � � � 1 � , s , � , i � � � | � � C 51 30 S-values Protein chain Protein chain restraint Violated if f � f o . f is the distance between two consecutive particles in a protein, f o is - - 1,680 the sum of the particle radii, and �� is 0.01 nm. Applied to particles: � � � , s , � , i � � � | � � 1 B � B j Z-axial position Violated for f < f o . f is the absolute Cartesian Z-coordinate of a protein particle, f o is the Immuno-Electron microscopy 456 lower bound defined for protein type � , and �� is 0.1 nm. Applied to particles: � � � � , s , � , i � � | � � 1, j � 1 B � B j - - 10,940 gold particles Violated for f > f o . f is the absolute Cartesian Z-coordinate of a protein particle, f o is the Protein localization restraint upper bound defined for protein type � , and �� is 0.1 nm. Applied to particles: 456 � � � � , s , � , i � � | � � 1, j � 1 B � B j Radial position Violated for f < f o . f is the radial distance between a protein particle and the Z-axis in a plane parallel to the X and Y axes, f o is its lower bound defined for protein type � , and 456 �� is 0.1 nm. Applied to particles: � � � � , s , � , i � � | � � 1, j � 1 B � B j - - Violated for f > f o . f is the radial distance between a protein particle and the Z-axis in a plane parallel to the X and Y axes, f o is its upper bound defined for protein type � , and 456 �� is 0.1 nm. Applied to particles: � � � , s , � , i � � � | � � 1, j � 1 B � B j Protein interaction restraint 13 contacts Protein contact Overlay assays Violated for f > f o . f is the distance between two protein particles, f o is the sum of the particle radii multiplied by a tolerance factor of 1.3, and �� is 0.01 nm. Applied to 20 112 20 particle: � � , s , � , i � � � � | � � (2,4,9), � � (1,2,3) B � B j Competitive binding restraint Protein contact 4 complexes Violated for f > f o . f is the distance between two protein particles, f o is the sum of the particle radii multiplied by a tolerance factor of 1.3, and �� is 0.01 nm. Applied to : Affinity purification 1 132 4 � � � � , s , � , i � � | � � (1,2,3), � � (2,4,6), � � ( Nup 82, Nic 96, Nup 49, Nup 57) B � B j Protein proximity restraint 64 complexes Protein proximity Violated for f > f o . f is the distance between two protein particles, f o is the maximal 692 25,348 692 diameter of a composite complex, and �� is 0.01 nm. Applied to particles: � � , s , � , i � � � � | � � (1,2,3), � � (2,4,9) B � B j

��������������������� �� Optimization Number of configurations 300 Contact similarity 0.60 200 0.52 100 0.44 0.36 0 0 2,000 4,000 10 10 10 8 10 6 10 4 10 2 0 Score Score

Integrating data a + + + Nuclear envelope Immuno-EM Ultracentrifugation Overlay assays pore volume Nucleoporin stoichiometry Affinity purifications NPC symmetry

��������������������� �� The STRUCTURE of NPC FG nucleoporins Outer rings Linker nucleoporins Inner rings Membrane rings Cytoplasm Nucleoplasm 5 nm Nup84 Nup82 Nic96 Nup133 Nup85 Sec13 Nup82 Spoke Nic96 Nup120 Nup145C Seh1 Pom152 Nup192 5 nm Nup157 Ndc1 Pom34 Nup188 Nup159 Nup170 Nup42 Nsp1 Nup59 Nup53 Nup100 Nup53 Nup59 Nup116 Nsp1 Nsp1 Nup49 Nup1 Nup145N Nup49 Nup57 Nsp1 Nup60 Nup57 Nup145N

IMP-based efforts TRiC/CCC Actin RyR channel Ribosomes, Hsp90 landscape Sali, Frydman, Chiu Sali, Chiu Sali, Serysheva, Chiu Sali, Frank; Sali, Akey Sali, Agard T Nuclear Pore Complex, Nup84 complex, Nuclear Pore Complex transport, Microtubule nucleation Sali, Rout, Chait Sali, Rout, Chait Sali, Rout, Chait, Chook, Liphardt Sali, Agard 26 Proteasome PCS9K-Fab complex Spindle Pole Body Chromatin globin domain Lymphoblastoid cell genome Sali, Baumeister Sali, Cheng, Agard, Pons Sali, Davis, Muller Marti-Renom Alber, Chen

Who Is developing with IMP?

From proteins to genomes Whale sperm myoglobin structure (1960) alpha-globin genomic domain structure (2011)