Embed Size (px)
Citation preview
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
https://cryoem.slac.stanford.edu/s2c2/training/workshops
NIH Funded National CryoEM Center
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
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
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
Gold-standard FSC (FSCg)
3.3Å
Estimating the Map Resolution
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
•
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
3.3 Å CryoEM Map of P22 Bacteriophage
Hryc, Chen et al PNAS 2017
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)
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)
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)
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)
3.9 Å 3.5 Å
Mohammad, Nature 2016 Roh, Mol Cell 2018
Compare FSC of Two Structures
Mohammad, Nature 2016 Roh, Mol Cell 2018
Roh, Mol Cell 2018
Features in the Extracellular Protein Subunits
Mohammad, Nature 2016
3.9 Å 3.5Å
Roh, Mol Cell 2018
Features in the Extracellular Protein Subunits
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
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.
