wah chiu, professor...lecture outline •de novo model building of a 3.3 Åmap •validation of...

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

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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

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0.9

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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


De Novo Model of Single Protein Subunit of P22 Phage


Full Atom Modeling of Single Protein Subunit of P22 Phage


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


0

0.1

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0.9

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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

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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°?


Mass-spec Identified the Assembly Factor Voa1



Function of Voa1

V0NDa

d

c”NT

f

C-Term Voa1 is a Component of the V0 Channel Rotor


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


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


2019 Model Challenge Targets

ADH 2.9 ÅAPOF 1.8 Å APOF 2.3 Å APOF 3.1 Å

EMD-20026additional map #1



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

wah chiu, professor...lecture outline •de novo model building of a 3.3 Åmap •validation of...

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