Detecting and Detecting and Characterizing Heterogeneity Characterizing Heterogeneity (Biochemical or Conformational) (Biochemical or Conformational) by Cryo-EM by Cryo-EM Eva Nogales HHMI / UC Berkeley LBNL
Seth Kostek Transcription Eukaryotic
CRSP ARC versus CRSP: Structure and Function Taatjes et al. (2002) Science 295
Dylan tj T Model of Action of CRSP and VP16 ARC
Dylan tj T Cofactor Flexibility
Spread, ill-defined variability 3-D variance and focused classification Pawel Penczek Patricia Grob Seth Kostek
3-D Variance and Supervised Classification: Principles Penczek Leschziner and Nogales (2007) Ann. Rev. Biophys. Bio. Struct. 3 Andres L h i
Structure and Flexibility of Human TFIID 3D Variance and Covariance Grob et al. (2006) Structure 14
Conformational Flexibility of Human TFIID Grob et al. (2006) Structure 14
Coordinated Motions in “Breathing” of TFIID Refined Grob et al. (2006) Structure 14
Human Pol II Conformational Sorting S Kostek et al. (2006) Structure, 14 Leschziner and Nogales (2007) Ann. Rev. Biophys. Patricia Bi l St t 3 G b
Localized variability of small but significant size Localized variability of small but significant size Cross-Correlation of Common Lines (CCCL) Cross-Correlation of Common Lines (CCCL) Richard Hall Bunpote Siridechadilok
Eukaryotic Translation Initiation: Cap-dependent and Cap-independent Pathways Bunpote Siridechadilok
Structure of Human eIF3 Random Conical Tilt Negative-stain Reference CRYO-EM DATA Siridechadilok et al. (2005) Science 310
Interaction with HCV IRES: The RNA is in Multiple Conformations Siridechadilok et al. (2005) Science 310
Testing the Principle of CCC with Synthetic Data Hall et al. (2007) 159 Bi l t J St
Comparison of Common Lines Hall et al. (2007) 159 Bi l t J St
3D Sorting and Statistics Number of projections Group 1 Group 2 Group 3 Conformation 1 100 35 13 Conformation 2 41 88 19 Conformation 3 22 17 110 Assignment 61% 63% 77% b Number of projections Group 1 Group 2 Group 3 Conformation 1 127 19 4 Conformation 2 7 137 6 Conformation 3 2 2 146 Assignment 93% 87% 94% Hall et al. (2007) J St t Bi l 159
Interaction with HCV IRES: The RNA is in Multiple Conformations Siridechadilok et al. (2005) Science 310
Interaction with the 40S Ribosome Christian Spahn, Kieft, Grassucci, Pencz Zhou, Doudna, and Frank (2001) Science 2 Siridechadilok et al. (2005) Science 310
Translation Initiation Interaction with eIF4G: Siridechadilok et al. (2005) Science 310 Model
Potentially large, widespread variability Potentially large, widespread variability requiring ab requiring ab initio initio separation separation Orthogonal Tilt Reconstruction (OTR) Orthogonal Tilt Reconstruction (OTR) Andres Leschziner
RCT followed by 3D MSA B. Sander, M.M. Golas and Holger Stark
The goal: a “ 90 o tilt” RCT and the “ Missing Cone” problem Orthogonal Tilt Reconstruction Random Conical Tilt (OTR) (RCT) “ Missing Cone” of views Andres L h i
OTR Reconstruction of the Chromatin Remodeling complex RS C Lid Lid Arm Base Central Arm Base Domain o 50A Leschziner et al. (2007) PNAS 104
Distinct Conformations of RS C “Open” “Closed” Conformation Conformation QuickTime™ and a Cinepak decompressor are needed to see this picture. QuickTime™ and a Cinepak decompressor are needed to see this picture. Leschziner et al. (2007) PNAS
The two maj or RS C conformers accommodate a nucleosome QuickTime™ and a QuickTime™ and a Cinepak decompressor Cinepak decompressor are needed to see this picture. are needed to see this picture. Leschziner et al. (2007) PNAS 104 Nucleosome (PDB:1AOI; Luger et al . 1997) filtered
Maximum Likelihood Maximum Likelihood
Leschziner and Nogales (2007) Ann. Rev. Biophys. Biomol. Struct.,3 Andres L h i
S.H. Scheres, H. Gao, M. Valle, G.T. Herman, PPB Eggermont, J. Frank, JM Carazo (2007) Nature Methods 4
Acknowledgements ARC/CRSP-Dylan Taajes Frank Andel Robert Tjian (UC Berkeley, (Boulder University) HHMI) TFIID-Patricia Grob Frank Andel Pawel Penczek (University of Texas) Carla Inouye / Robert Tjian PolII - Seth Kostek Sacha de Carlo, Jie Fang Patricia Grob RSC - *Andres Leschziner Anjanabha Saha / Brad Cairns (University of Utah, HHMI) (Harvard University) eIF3 - Richard Hall *Bunpote Siridechadilok Chris Fraser / Jennifer Agouron, DOE, HHMI, NCI, NIH Doudna (UC Berkeley, HHMI)
20-fold symmetry 7-fold symmetry ASYMMETRIC!
Putting it all together Pol II CRSP ARC TFIID PBAF Activators TFIIA-B-D CTD TRANSCRIPTOSOME + Nucleosome
How do t his new reconst ruct ion compare wit h t he previously published ones? RSC RSC PBAF (RCT) (OTR) (RCT) Asturias et al . (2002) Leschziner et al . (2005) PNAS 99 : 13,477 S t ructure 13 :267
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