Wah Chiu Baylor College of Medicine wah@bcm.edu - - PowerPoint PPT Presentation

Wah Chiu Baylor College of Medicine wah@bcm.edu

http://ncmi.bcm.edu

Research Missions at NCMI

• Develop and apply Cryo-EM for structure

determinations of Molecular Nano-Machines in solution states towards atomic resolution

• Share our experimental and computational

technology freely with the global academic community

Electron Cryo-Microscope at NCMI, Baylor College of Medicine

200kV 200kV 300kV 300kV

data collection image processing reconstruction structural analysis Model; Deposition imaging cryo-em sample preparation biochemical preparation

Pipeline in Biological Cryo-EM

Cryo-EM: A Critical Tool in Biomedicine

• Can visualize bio-structures at a broad range of

resolutions and complexities

200 nm 0.4nm

Optical microscopy Electron cryotomography Electron cryomicroscopy X-ray microscopy Electron cryotomography Electron & X-ray Crystallography NMR

50nm 8nm 5nm 0.9nm 0.6nm 0.1nm

Crystallography

0.3nm

Structural Biology from Cells to Atoms

Electron cryomicroscopy

Trends in Macromolecular Cryo-EM

Matthew Baker (2007)

50 100 150 200 250

Number of Publications

1990 1992 1994 1996 1998 2000 2002 2004 2006

Year

CryoEM Maps in EBI EMDB

20 40 60 80 100 120 140 2002 2003 2004 2005 2006 2007 Y ear Num ber of Structures

Cryo-EM: A Critical Tool in Biomedicine

• Can visualize bio-structures at a broad range of

resolutions and complexities

• Is the only method to look at structures of

certain molecular machines

calmodulin scruin actin 1:1:1 102kDa 42kDa 24kDa Cryo-EM image

3 filaments

Schmid, Sherman, Matsudaira, Chiu (2004) Nature, 431: 104-107

Cong, Topf, Sali, Matsudaira, Chiu, Schmid (2007) JMB, in press

S E

Actin Scruin

Cryo-EM: A Critical Tool in Biomedicine

• Can visualize bio-structures at a broad range of

resolutions and complexities

• Is the only method to determine structures of

certain molecular machines

• Can do de novo Cα backbone trace without

crystallography

JEM3200 (Yoshi type) 300kV FEG Liquid helium 4k Gatan CCD

Imaging Epsilon15 Phage at Liquid He

JEM3000 300kV 4°K 60Kx mag ~28 e/Å2 Film data J Jakana

Chen, Jakana and Chiu (2007). J Chinese Elec Microsc 26: 473-479.

Computed FFT of Images

F2(s) CTF2(s) Env2(s) + N2(s)

Structure factor Contrast transfer function Envelope function Background

SNR (Contrast) = (F2 CTF2 Env2 ) / N2

Env2(s) ~ exp (-2BS2)

Saad etal (2001) J Struct Biol 133: 32-42

Epsilon15 Image Data Recorded with Liquid Helium for 4.5 Å Map

• 3,000 micrographs were digitized
• 40% has SNR beyond 6 Å
• Images with non-isotropic CTF were discarded
• 36,000 particles were picked from 1,228

micrographs

• 20,000 particles were finally used for 3-D

reconstruction

Jiang, Baker, Jakana, Weigele, King, Chiu (unpublished)

Cryo-EM: A Critical Tool in Biomedicine

• Can visualize bio-structures at a broad range of

resolutions and complexities

• Is the only method to look at structures of

certain molecular machines

• Can do de novo Cα backbone without

crystallography

• Can determine subnanometer resolution

structure with few tens to thousands of particle images

Zhou, Baker, Jiang, Dougherty, Jakana, Dong, Lu and Chiu (2001) Nature SB. 8: 868-73

Liu, Jiang, Jakana and Chiu (2007) JSB 160:11-27

7.9 Å cryoEM map of Rice Dwarf Virus Reconstructed from 284 Particles

Multi-Path Monte Carlo Simulated Annealing Optimization Algorithm

41, 9.8Å, 1α, 0β 62, 9.6Å, 4α, 0β 81, 9.2Å, 10α, 0β 101, 8.9Å, 14α, 2β

Cryo-EM Maps (numbers of particles)

128, 8.6Å, 12α, 3β 147, 8.3Å, 15α, 3β 181, 8.3Å, 16α, 4β 284, 7.9Å, 20α, 7β

Liu, Jiang, Jakana and Chiu (2007) JSB 160:11-27.

(a) (b) (c)

Reconstructions from Various Subsets of 284 Particle Images

(d)

Liu, Jiang, Jakana and Chiu (2007) JSB 160:11-27

Cryo-EM: A Critical Tool in Biomedicine

• Can visualize bio-structures at a broad range of

resolutions and complexities

• Is the only method to look at structures of

certain molecular machines

• Can do de novo Cα backbone without

crystallography

• Can determine subnanometer resolution

structure with few tens to thousands of particle images

• Can detect protein conformational changes in a

physiological process

500Å

Procapsid shell Diameter = 585 Å Mature phage Diameter = 700 Å

Electron Images of P22 Phage

Jiang et al (2003) Nat Struct Biol 10: 131-135.

Procapsid Mature phage

Large Structural Changes in P22 Maturation

Cryo-EM: A Critical Tool in Biomedicine

• Can visualize bio-structures at a broad range of

resolutions and complexities

• Is the only method to look at structures of certain

molecular machines

• Can do de novo Cα backbone without crystallography
• Can determine subnanometer resolution structure with

few tens to thousands of particle images

• Can detect protein conformational changes in a

physiological process

• Can provide a key data set for computational biology

research to extract additional stuctural information

Structure & Function Discovery Crystallography NMR Cryo-EM Microscopy FRET Simulations Modeling Bioinformatics Mass Spectroscopy Proteomics

Data Integration

2a 2b 1 9a 9b 10 6 4 8a 3a3b 5 2a 3c 7a 7b 8b 11 12 TM 5 6 8a 4 3a 3b 9a 9b 10

9.6 Å Cryo-EM Map of RyR1 (2.2 MDa)

Compare the pore in RyR1 and K+ channels

Helix 2 Helix 1

RyR1 MthK

Pore helix Inner helix Filter Pore helix Inner helix Filter

KcsA

lumenal side (‘out’) kink cytoplasmic side (‘in’)

Helix 1 (inner) Helix 2 (pore)

Ludtke, Serysheva et al Structure (2005)

Sequence assignment of observed helices

4864 –NKSEDEDEPDMKCDDMMTCYLFHMYVGVRAGGGIGDEIEDPAGDEYELYRVVFDITFFFFVIVILLAIIQGLIIDAFGELRDQQEQVKEDMETK- 4957

***** * ** ** ** ** * * * * * * * *

Filter M9 M10

RyR1:

Helix 2 (M4879-E4893) Pore helix Inner helix

45 -SWTVSLYWTFVTIATVGYGDYSPSTPLGMYFTVTLIVLGIGTFAVAVERLLEFLINREQ- 103

Hinge

MthK:

Hinge

(I4918-E4948)

Helix 1

1 5037

4864 4967

Highly conserved region (>90% identity) among RyRs Mutations within these regions of RyRs (G4895A, I4898A,

D4900N) alter rates of ion translocation

Sequence Assignment of Observed α- Helices in the TM Region

Zorzato et al 1990

Filter

Target sequence Target sequence Template sequence Template structure Template sequence Template structure CryoEM density segment CryoEM density segment

(Modeller, Mod-EM, Moulder)

CryoEM density constrained modeling

CryoEM Restrained Comparative Modeling

Sequence Sequence Target identification CryoEM Density CryoEM Density Model localization Structural template Structural template

(Blast, Psi-blast)

Model (threading) Model (threading)

(Modeller)

Fitted model Fitted model

(Foldhunter, Situs)

CryoEM density segment CryoEM density segment

(Manual Segmentation)

Multiple sequence alignments Multiple sequence alignments

+

Multiple Models Multiple Models Scored models Scored models Selected model Selected model Fitted models Fitted models Evolve best alignment Evolve best alignment (25x) Final model Final model

Challenges and Opportunities

• Specimens with conformational variability
• 2-3 Å map of single particles
• Integrate with other information for knowledge

discovery

• Extend post-averaging of cryoET sub-

tomograms to molecular resolution

• Engage cryoEM study to translational medicine
• Relatively high cost of very high-end

instruments (development, acquisition and maintenance)