Detecting and Detecting and Characterizing Heterogeneity - - PowerPoint PPT Presentation

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Detecting and Detecting and Characterizing Heterogeneity - - PowerPoint PPT Presentation

Feb 20, 2023 135 likes •497 views

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

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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

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SLIDE 2

Eukaryotic Transcription

Seth Kostek

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SLIDE 3

ARC versus CRSP: Structure and Function

Taatjes et al. (2002) Science 295

CRSP

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SLIDE 4

Model of Action of CRSP and ARC

VP16

Dylan T tj

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SLIDE 5

Cofactor Flexibility

Dylan T tj

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SLIDE 6

Spread, ill-defined variability 3-D variance and focused classification

Pawel Penczek Patricia Grob Seth Kostek

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SLIDE 7

3-D Variance and Supervised Classification: Principles

Leschziner and Nogales (2007)

  • Ann. Rev. Biophys. Bio. Struct. 3

Penczek

Andres L h i

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SLIDE 8

Structure and Flexibility of Human TFIID

Grob et al. (2006) Structure 14

3D Variance and Covariance

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SLIDE 9

Conformational Flexibility of Human TFIID

Grob et al. (2006) Structure 14

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SLIDE 10

Coordinated Motions in “Breathing”

  • f TFIID

Refined

Grob et al. (2006) Structure 14

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SLIDE 11

Human Pol II Conformational Sorting S

Kostek et al. (2006) Structure, 14 Leschziner and Nogales (2007) Ann. Rev. Biophys. Bi l St t 3

Patricia G b

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SLIDE 12

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

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SLIDE 13

Eukaryotic Translation Initiation: Cap-dependent and Cap-independent Pathways

Bunpote Siridechadilok

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SLIDE 14

Structure of Human eIF3

Random Conical Tilt Negative-stain Reference CRYO-EM DATA

Siridechadilok et al. (2005) Science 310

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SLIDE 15

Interaction with HCV IRES: The RNA is in Multiple Conformations

Siridechadilok et al. (2005) Science 310

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SLIDE 16

Testing the Principle of CCC with Synthetic Data

Hall et al. (2007) J St t Bi l 159

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SLIDE 17

Comparison of Common Lines

Hall et al. (2007) J St t Bi l 159

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SLIDE 18

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%

3D Sorting and Statistics

Hall et al. (2007) J St t Bi l 159

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SLIDE 19

Interaction with HCV IRES: The RNA is in Multiple Conformations

Siridechadilok et al. (2005) Science 310

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SLIDE 20

Interaction with the 40S Ribosome

Christian Spahn, Kieft, Grassucci, Pencz Zhou, Doudna, and Frank (2001) Science 2

Siridechadilok et al. (2005) Science 310

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Interaction with eIF4G: Translation Initiation Model

Siridechadilok et al. (2005) Science 310

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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

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SLIDE 23

RCT followed by 3D MSA

  • B. Sander, M.M. Golas and Holger

Stark

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The goal: a “ 90o tilt”

Random Conical Tilt (RCT) Orthogonal Tilt Reconstruction (OTR) “ Missing Cone”

  • f views

RCT and the “ Missing Cone” problem

Andres L h i

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SLIDE 25

50A

  • Lid

Arm Arm Base Central Domain Lid Base

OTR Reconstruction of the Chromatin Remodeling complex RS C

Leschziner et al. (2007) PNAS 104

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Distinct Conformations of RS C

Leschziner et al. (2007) PNAS

QuickTime™ and a Cinepak decompressor are needed to see this picture.

QuickTime™ and a Cinepak decompressor are needed to see this picture.

“Open” Conformation “Closed” Conformation

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The two maj or RS C conformers accommodate a nucleosome

Nucleosome (PDB:1AOI; Luger et al. 1997) filtered

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 104

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Maximum Likelihood Maximum Likelihood

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SLIDE 29

Andres L h i

Leschziner and Nogales (2007) Ann. Rev. Biophys. Biomol. Struct.,3

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SLIDE 30

S.H. Scheres, H. Gao, M. Valle, G.T. Herman, PPB Eggermont,

  • J. Frank, JM Carazo (2007) Nature Methods 4
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SLIDE 31

ARC/CRSP-Dylan Taajes Frank Andel (Boulder University) Robert Tjian (UC Berkeley, 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 Doudna (UC Berkeley, HHMI)

Acknowledgements

Agouron, DOE, HHMI, NCI, NIH

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20-fold symmetry 7-fold symmetry ASYMMETRIC!

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Putting it all together

TRANSCRIPTOSOME

TFIIA-B-D

Activators CTD + Nucleosome

TFIID Pol II CRSP ARC PBAF

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SLIDE 34

Asturias et al. (2002) PNAS99: 13,477 Leschziner et al. (2005) S t ructure 13:267

RSC (RCT) RSC (OTR) PBAF (RCT)

How do t his new reconst ruct ion compare wit h t he previously published ones?