wah chiu, ph.d. professor department of bioengineering and ... · references •frank, j. (2006)....
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Wah Chiu, Ph.D.
ProfessorDepartment of Bioengineering and
Department of Microbiology and Immunology, Stanford University
DirectorDivision of CryoEM and Bioimaging, SSRL,
SLAC National Accelerator Laboratory, Stanford University
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https://cryoem.slac.stanford.edu/s2c2/training/workshops
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NIH Funded National CryoEM Center
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S2C2 at the Arrillaga Science Center, SLAC
TEM Rooms (4 total) – 1,350 SF
Equipment Rooms – 560 SF
Control Room/User space – 870 SF
Low Humidity Room – 325 SF
Biochem Wet Lab (shared) – 3,000 SF
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Direct Electron Detector (DE20) Image of P22 Bacteriophages Embedded in Ice
D Chen & J Jakana
• 300 keV JEM3200FSC
• 0.85 μm defocus
• 1.07Å/pixel
• 1.56e/Å2/frame
• Motion corrected and damage weighted
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2 Independent Maps from 2 Data Subsets and 2 Initial Models
Subset 1, N=11,000
Subset 2, N=11,000
Map 1
Map 2Liu, X, Jiang, W, (2007) JSB; Guo, F, Jiang, W, (2014) Meth Mol Bio
EMAN2, MPSA, JSPR Hryc, Chen et al PNAS 2017
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Gold-standard FSC (FSCg)
3.3Å
Estimating the Map Resolution
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Validation of CryoEM Map
Assure No Over-Refinement: Randomize the phases of all the raw particle images for frequencies beyond 75% of the targeted resolution
•
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Estimate the Map Resolution with 2 Maps
4.5Å-phase-randomized FSC (FSCn)
Gold-standard FSC (FSCg)
True FSC (FSCt)
0.143
4.3Å
4.5Å3.3Å
c
Hryc, Chen et al PNAS 2017
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3.3 Å CryoEM Map of P22 Bacteriophage
Hryc, Chen et al PNAS 2017
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50 nm50 nm
a b
d
3 Å
c
Uncorrected Drift and radiation damage corrected
Cryo-EM of GroEL (~1MDa)
2D Class averages
JEM3200FSC; K2 camera; 36,000 particles
SH Roh et al PNAS (2017)
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N-Terminus
C-Terminus
LYS4
VAL6PHE8
VAL521 THR517
ARG13
ARG18 LYS15
LEU17
VAL22
ASN21
TYR506SER509
ALA508 LEU504
SER502
GLN505
LEU215 VAL213
ILE325VAL323
PHE195
MET193
ILE332
THR330
3.5 Å Cryo-EM Structure of GroEL
Top
Side
SH Roh et al PNAS (2017)
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Spatial Frequency (1/Å)
Fou
rier
Sh
ell C
orr
elat
ion
3.5Å0.143
0.5
Resolution Estimation
SH Roh et al PNAS (2017)
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Resmap
Apical Domain Has Lower Resolvability and Positional Accuracy
Crystal structure(PDB 1SS8)
Cryo-EM Model
Atomic displacement parameter (ADP)
150
115
80
150
75
0
SH Roh et al PNAS (2017)
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3.9 Å 3.5 Å
Mohammad, Nature 2016 Roh, Mol Cell 2018
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Compare FSC of Two Structures
Mohammad, Nature 2016 Roh, Mol Cell 2018
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Roh, Mol Cell 2018
Features in the Extracellular Protein Subunits
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Mohammad, Nature 2016
3.9 Å 3.5Å
Roh, Mol Cell 2018
Features in the Extracellular Protein Subunits
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Roh, Mol Cell 2018
3.9 Å
3.5Å
TMHs Interacting with the protein
TMHs Exposed to membrane
TMHs Exposed to membrane
Lipid-Exposed TM Subunits
Mohammad, Nature 2016
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References• Frank, J. (2006). Three-dimensional electron microscopy of macromolecular assemblies :
visualization of biological molecules in their native state. New York, Oxford University Press.
• Glaeser, R. M., K. H. Downing, D. DeRosier, W. Chiu and J. Frank (2007). Electron crystallography of biological macromolecules. Oxford ; New York, Oxford University Press.
• Rosenthal, P. B. and R. Henderson (2003). "Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy." J Mol Biol 333(4): 721-745.
• Chen, S., G. McMullan, A. R. Faruqi, G. N. Murshudov, J. M. Short, S. H. Scheres and R. Henderson (2013). "High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy." Ultramicroscopy 135: 24-35. PMC3834153.
• Hryc, C. F., D. H. Chen, P. V. Afonine, J. Jakana, Z. Wang, C. Haase-Pettingell, W. Jiang, P. D. Adams, J. A. King, M. F. Schmid and W. Chiu (2017). "Accurate model annotation of a near-atomic resolution cryo-EM map." Proc Natl Acad Sci U S A 114(12): 3103-3108. PMC5373346.