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NIH U24GM129564
Wah Chiu, Professor
√
https://commonfund.nih.gov/CryoEM
Created with funding from the NIH Common Fund –the three Centers interact and coordinate activities
The Cryo-EM National Centers
https://cryoem.slac.stanford.edu/s2c2/
SLAC National Accelerator Laboratory, Stanford University,
Menlo Park, CA
PI: Wah Chiu
Oregon Health and ScienceUniversity and Pacific Northwest
National Laboratory, Portland, OR
https://pncc.labworks.org/
PI: Eric Gouaux
http://nccat.nysbc.org/
New York Structural Biology Center, New York, NY
PI: Bridget Carragher
National Center for CryoEM Access and Training
Cryo-EM Statistics
Lecture Outline
• De novo model building of a 3.3 Å map
• Validation of model with mass spectroscopy of a 3.5 Å map
• Detection of water and their validation of a 2.7 Å map
• Structure details of a 1.34 Å map
• Cryo-EM structure Challenge with the community
Direct Electron Detector (DE20) Image of P22 Phages
D Chen & J Jakana, 2015
• 300 keVJEM3200FSC
• 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 2
EMAN2, MPSA, JSPR Hryc, Chen et al PNAS 2017
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Four
ier S
hell
Corr
elat
ion
Frequency (1/Å)
Gold-standard FSC (FSCg)
3.3Å
FSC =(F1å • F2* )F1
2 • F22
Estimate CryoEM Map Resolution with 2 Independent Maps
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Four
ier S
hell
Corr
elat
ion
Frequency (1/Å)
4.5Å-phase-randomized FSC (FSCn)
Gold-standard FSC (FSCg)
True FSC (FSCt)
0.143
4.3Å4.5Å
3.3Å
c
Hryc, Chen et al PNAS 2017
FSC =(F1å • F2* )F1
2 • F22
Phase Randomization of Images at ¾ of Claimed resolution
3.3 Å Cryo-EM Map of P22 Bacteriophage
Hryc, Chen et al PNAS 2017
De Novo Model of Single Protein Subunit of P22 Phage
Hryc, Chen et al PNAS 2017
Full Atom Modeling of Single Protein Subunit of P22 Phage
Hryc, Chen et al PNAS 2017
THR98
ARG102
TYR100 ARG103
ARG109
LEU111
ASN113 GLU116
LYS118
MET122
ALA120
MET126
Long Helix
LYS377THR379
PHE392
THR394
PHE381
ASP83ASP85
PHE87
LEU409
SER382
P-Domain
MET49
PRO50
TRP48
ILE47
ARG42
GLN41
PRO36TYR33
F-Loop
TRP61
GLU59
GLN58
ASP56
THR68
THR64
ASP62
GLU54
E-Loop
A-Domain
GLN173
TYR175
GLY179
TYR177 TYR180
LEU182
LYS184
Structural Details of P22 Phage Capsid Protein
Hryc, Chen et al PNAS 2017
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5Fo
urie
r She
ll Co
rrel
atio
n
Spatial Frequency (1/Å)
Even Map vs. EvenModel
3.4Å0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5Fo
urie
r She
ll Co
rrel
atio
n
Spatial Frequency (1/Å)
Even Map vs. EvenModel
Odd Map vs. EvenModel
3.4Å
Model Validation: 2 Independent Maps
Map 1 (Even)
Map 2 (Odd) Hryc, Chen et al PNAS 2017
Penton
Hexon
Models of 7 Subunits in an AU of P22 Phage A B
C
18.2Å 0 ÅD
Hryc et al PNAS 2017
Fit-to-density for an Asymmetric Unit (7 individual subunits)
Cross-correlation: 0.836 EMRinger Score: 3.58
MolProbity (of an asymmetric unit)
All-Atom ContactsClashscore, All Atoms 10.26 97th percentile * (N=37, 3Å-9999Å)
Clashscore is the number of steric overlaps (>0.4Å) per 1000 atoms.
Protein Geometry
Poor rotamers 0 0.00% Goal: 0.25Å 0 0.00% Goal: 0%
Bad backbone bonds: 0/23401 0.00% Goal: 0%
Bad backbone angles: 4/31815 0.01% Goal:
Map-Derived Model
Experimental Map
Representation of Cryo-EM Map & Model
Calculated Map from Model
Apply Weights
Map-Derived Model
Weighted Map
Calculated Map from Model
25 Å2115ADP
Interactions Between Adjacent Subunits Within a Hexon or a Penton Via Salt Bridges
8 interactions per adjacent subunits Hryc, Chen et al PNAS 2017
CryoEM Image of Vo Channel (330 kDa) Biochemically Extracted from Yeast Vacuole
Osteoporosis/petrosisRenal tubular acidosisDiabetesNeurodegenerationPathogen entryCancer
pH HomeostasisDevelopmentEndocytosismTOR signalingNotch signalingProtein sorting/trafficking
150,000 channel particles recorded in JEM3200 and K2Roh, Wilkens et al. Mol Cell 2018
V1 Motor - ATPase
VO Proton Channel Mobile Rotor
3.5 Å Cryo-EM Map of Vo Channel (330 kDa)
Roh, Wilkens et al. 2018 Mol Cell
180°
Cytoplasm
Lumen
Rotor (c-ring)
Proton channel (a)
Connecting adaptor (d)
Full Atomic Models for 14 Protein Subunits of Vo Channel
Accessary (e/f)
Roh, Wilkens et al. Mol Cell 2018
Helix with an Unknown Biochemical Identity in V0 Channel
90°?
Roh, Wilkens et al. Mol Cell 2018
Mass-spec Identified the Assembly Factor Voa1
Roh, Wilkens et al. Mol Cell 2018
Roh, Wilkens et al. Mol Cell 2018
Function of Voa1
V0NDa
d
c”NT
f
C-Term Voa1 is a Component of the V0 Channel Rotor
Roh, Wilkens et al. Mol Cell 2018
V0ND (voa1△)a
d
c”NT
f
2.7 Å Cryo-EM Structure of V0 Channel
Roh, Wilkens et al. Science Adv (2020) accepted
Validation of Water in the Channel with MD Simulation
Roh, Wilkens et al. Science Adv (2020) acceptedWater Finders program (Coot); Q score (Pintilie, Nat Methods, 2020)Distance between H20 and O and N atoms
Validation of Water in the Channel Bridging Q80 Sidechain Nitrogen and V7 Carbonyl Oxygen in 10 c-Ring proteins
Roh, Wilkens et al. Science Adv (2020) accepted
1.34 Å Cryo-EM Map of Apoferritin (Krios-BioQ-K3)
(Å)
-0.2
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1Spatial Frequency (1/Å)
Four
ierS
hell
Cor
rela
tion
(FSC
)
0.143 FSC 1.34 Å
200 Å
6
7
8
9
10
11
12
13
14
0.2 0.3 0.4 0.5
Ln(N
umbe
r of
Par
ticle
s)
1/Resolution2 (Å-2)
Particle Number VS Resolution
Kaiming Zhang, Stanford, unpublished
FSC =(F1å • F2* )F1
2 • F22
~900,000 ParticlesRelion
Rosenthal &Henderson (2003) JMB
Side Chain Densities of 1.34 Å Cryo-EM Map of Apoferritin
His118
Asp126
Phe51
Asn25 Val33
Met158 Lys108
Arg9
ChargedHydrophobicPolar
Thr153
Gln73 Ile80
Pro127
Glu167
Cys130
Leu155
Trp93
Ala52
Gly149Ser31
Tyr32
Side chains with alternate conformations
Glu64
a b
Gln14 Cys102Lys49 Lys53
a b a bab
ca b a b
Glu61 Glu94
a b
Arg156
a b
Asn Val
Pintilie, Li, Zhang et al. (unpublished)
Water Finder in 1.34 Å Cryo-EM Map of Apoferritin
05101520253035404550
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0
H2O-NH2O-O
Distance (Å)
Waters found in the 1.34 ÅcryoEM Map with
Water Finder232
Water Molecules in X-ray mapPDB:3ajo. 229
Pintilie, Li, Zhang et al. (unpublished)
05101520253035404550
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0
H2O-NH2O-O
Distance (Å)
Waters found in the 1.34 ÅcryoEM Map with
Water Finder. 232
Water Finder in cryoEM mapPDB:3ajo in Xray map
Water Finder in 1.34 Å Cryo-EM Map of Apoferritin
Pintilie, Li, Zhang et al. (unpublished)
2019 Model Challenge Targets
ADH 2.9 ÅAPOF 1.8 Å APOF 2.3 Å APOF 3.1 Å
EMD-20026additional map #1
EMD-20027additional map #2
EMD-20028additional map #2
EMD-0406primary map
BIORXIV/2020/147033
2019 Challenge: 4 Metrics Stood Out
CaBLAM: virtual dihedrals based on Cɑ, C=O compared to statistics from high quality PDB models [Williams 2018]
Coordinates alone:
MAPQ Q-score: Per-atom Correlation vs Reference Gaussian (r=0-2 Å) [Pintilie 2020]
PHENIX Resolution Map-Model FSC = 0.5 [Afonine 2018]
EMRinger Z-score “rotamericity” of map for protein Cgatom paths around c1 [Barad 2015]
Fit-to-Map:
BIORXIV/2020/147033
2019 Model Challenge Results
AcknowledgementsM.I.T.: Cammie Haase-Pettingell, Jon King
SUNY: Nicholas Stam, Stephan Wilkens
Arizona State University/University of Illinois: Mrinal Shekhar, Chris Chipot, Abhishek Singharoy
Rutgers University: Catherine Lawson, Helen Berman
UC Davis: Andriy Kryshtafovych
Chiu lab
Corey Hryc (Industry)
DongHua Chen (Stanford U)
Soung Hun Roh (National Seoul U)
Greg Pintilie (Stanford U)
Kaiming Zhang (Stanford U)
Shanshan Li (Stanford U)
Michael Schmid (Stanford U)
NIGMS
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