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
Baylor College of Medicine Electron Cryo-Microscope 300kV at NCMI, 300kV 200kV 200kV
Pipeline in Biological Cryo-EM cryo-em sample biochemical preparation preparation imaging data collection image processing Model; reconstruction structural analysis Deposition
Cryo-EM: A Critical Tool in Biomedicine • Can visualize bio-structures at a broad range of resolutions and complexities
Structural Biology from Cells to Atoms Optical Electron Electron Crystallography microscopy cryotomography cryomicroscopy 0.1nm 8nm 0.9nm 0.4nm 200 nm 50nm 5nm 0.6nm 0.3nm X-ray microscopy Electron Electron Electron & X-ray cryotomography cryomicroscopy Crystallography NMR
Trends in Macromolecular Cryo-EM 250 200 Number of Publications 150 100 50 0 1990 1992 1994 1996 1998 2000 2002 2004 2006 Year Matthew Baker (2007)
CryoEM Maps in EBI EMDB 140 120 100 Num ber of Structures 80 60 40 20 0 2002 2003 2004 2005 2006 2007 Y ear
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
Cryo-EM image scruin 102kDa actin 42kDa calmodulin 24kDa 1:1:1
3 filaments Schmid, Sherman, Matsudaira, Chiu (2004) Nature , 431 : 104-107
Cong, Topf, Sali, Matsudaira, Chiu, Schmid (2007) JMB , in press
Scruin Actin E S
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 C α 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/Å 2 Film data J Jakana Chen, Jakana and Chiu (2007). J Chinese Elec Microsc 26 : 473-479.
Computed FFT of Images F 2 (s) CTF 2 (s) Env 2 (s) + N 2 (s) Structure factor Envelope function Background Contrast transfer function SNR (Contrast) = (F 2 CTF 2 Env 2 ) / N 2 Env 2 (s) ~ exp (-2BS 2 ) 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 C α 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
7.9 Å cryoEM map of Rice Dwarf Virus Reconstructed from 284 Particles Multi-Path Monte Carlo Simulated Annealing Optimization Algorithm Liu, Jiang, Jakana and Chiu (2007) JSB 160 :11-27
Cryo-EM Maps (numbers of particles) 41, 9.8Å, 1 α , 0 β 62, 9.6Å, 4 α , 0 β 81, 9.2Å, 10 α , 0 β 101, 8.9Å, 14 α , 2 β 128, 8.6Å, 12 α , 3 β 147, 8.3Å, 15 α , 3 β 181, 8.3Å, 16 α , 4 β 284, 7.9Å, 20 α , 7 β Liu, Jiang, Jakana and Chiu (2007) JSB 160 :11-27.
Reconstructions from Various Subsets of 284 Particle Images (a) (b) (c) (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 C α 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
Electron Images of P22 Phage 500Å Procapsid shell Mature phage Diameter = 585 Å Diameter = 700 Å
Jiang et al (2003) Nat Struct Biol 10 : 131-135.
Large Structural Changes in P22 Maturation Procapsid Mature phage
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 C α 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
Data Integration Cryo-EM Modeling Crystallography Structure & Simulations NMR Function Discovery Mass Bioinformatics Spectroscopy Proteomics Microscopy FRET
9.6 Å Cryo-EM Map of RyR1 (2.2 MDa) 6 8a 11 4 TM 3b 3c 2a 12 3a 5 9a 7a 7b 9b 8b 10 6 8a 1 4 3a3b 2a 2b 5 9a 9b 10
Compare the pore in RyR1 and K + channels KcsA MthK RyR1 Pore helix Filter Filter Pore helix Helix 2 Inner helix Inner helix Helix 1 lumenal side (‘out’) Helix 2 (pore) kink Helix 1 (inner) Ludtke, Serysheva et al cytoplasmic side (‘in’) Structure (2005)
Sequence Assignment of Observed α - Sequence assignment of observed helices Helices in the TM Region 1 5037 4864 4967 Hinge Filter RyR1 : 4864 –NKSEDEDEPDMKCDDMMTCYLFHMYVGVRAGGGIGDEIEDPAGDEYELYRVVFDITFFFFVIVILLAIIQGLIIDAF G ELRDQQEQVKEDMETK- 4957 ***** * ** ** ** ** * * * * * * * * M9 M10 Zorzato et al (I4918-E4948) 1990 Helix 1 Helix 2 (M4879-E4893) Filter Hinge MthK : 45 -SWTVSLYWTFVTIATVGYGDYSPSTPLGMYFTVTLIVLGIGTFAVAVERLLEFLINREQ- 103 Pore helix Inner helix � Highly conserved region (>90% identity) among RyRs � Mutations within these regions of RyRs (G4895A, I4898A, D4900N) alter rates of ion translocation
CryoEM Restrained Comparative Modeling Target identification (Blast, Psi-blast) (Modeller) Sequence Structural template Model (threading) Sequence Structural template Model (threading) Model localization CryoEM density CryoEM density (Foldhunter, Situs) (Manual Segmentation) CryoEM Density Fitted model CryoEM Density Fitted model segment segment Target Template sequence Multiple sequence alignments Target Template sequence Multiple sequence alignments + sequence Template structure sequence Template structure Multiple Models Multiple Models Evolve best Evolve best CryoEM density segment Fitted models CryoEM density segment Fitted models alignment alignment Scored models Scored models (25x) CryoEM density constrained modeling Selected model Selected model Final model Final model (Modeller, Mod-EM, Moulder)
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)
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