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Molecular Electron Microscopy Cryo-Electron Microscopy

James Conway BST-3, Room 2047 jxc100@pitt.edu 412-383-9847

U. Pitt. MBSB-1 Fall-2014

Friday: Introduction to Molecular Electron Microscopy Monday: Single-Particle EM Monday: High Resolution and Labeling

Methods, Samples & Symmetry

• 2D crystallography membrane proteins: bacteriorhodopsin, LHC-II, aquaporin other proteins: tubulin

• Helical structures microtubules, bacterial flagellar filament

filamentous phages, filoviridae (eg, Ebola) • Particles

Icosahedral virus capsids/complexes Lower symmetry Clp-family of ATP-dependent proteases No symmetry ribosome, phage particles

• Non-uniform structures

Tomography viruses, organelles, cells

Image Reconstruction

• So we have some electron micrographs, what’s next? • Image reconstruction: calculate a 3D density map • Raw data are 2D projection images • We can think of a projection image as a 2D density function

representing a 3D volume • Use the projection direction (orientation) for each image in a

set images, back-project to simulate the original 3D volume • “Single particle” and “tomography” are essentially the same:

- SPR: many objects, orientations unknown - Tomo: one object, orientations ~known

Metal Shadow Negative-Staining CryoEM

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Reconstructions - Central Section Theorem!

Projection onto 2D plane (microscopy)

Fourier

Transform

Fit through center of 3D volume

Fourier

Transform (inverse)

center of Fourier plane

center of Fourier volume

Reconstruction pathways

Need many images at different orientations to fill Fourier volume or Real Space volume

Raw images

Object!

Back-Projection in 2D - 1. Projection images!

Object!

Back-Projection in 2D - 2. Projecting backwards!

Object!

Back-Projection in 2D - 3. Finer sampling!

10¡ ! 5¡ ! 1¡ !30¡ !

3

Object!

Back-Projection in 2D - 4. Details!

10¡ !1¡ !

1¡ !R-weighted!

Object!

Back-Projection in 2D - 5. Another Example!

1¡ !

R-weighted!

Reconstructions - Central Section Theorem!

Projection onto 2D plane (microscopy)

Fourier

Transform

Fit through center of 3D volume

Fourier

Transform (inverse)

center of Fourier plane

center of Fourier volume

Reconstruction pathways

Need many images at different orientations to fill Fourier volume or Real Space volume

Raw images

Analysis of Single Particles

Single Particles means many isolated, or free-standing particles and preferably random views

Combine different views of identical objects

Resolution will be limited by: - degree of identity - accuracy of view estimations - completeness of dataset (views) - S/N and amount of data - everything else!

Symmetry & size help, eg, icosahedral virus capsids

Contrast helps, eg cryo-negative stain

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SPO1"T=16, 950-1080• !

5! 5!6! 6!6! 6! 6!6!5! 5!

5! 5!

6!6!

Capsids!

HBV"T=4, 350• !

5! 5!

6!6!

6!

T5"T=13l, ~880• !

Herpesvirus"T=16, 1250• !HK97"

T=7l, 480/600 • !, 480/600 •

5! 5!6!

6!

5 5

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Capsids

5

, 480/600 •, 480/600 •

5! 5!

6!6! 6!

6!6!6!

6!

Gifsy2!T=7l, 600 • !

Phage G"T=52, 1500-1800 • !

5! 5!6!

Adenovirus 3 Dodecahedron!DB: 250• ! DF: 400• !

!

T-numbers

The icosahedron has 60-fold symmetry: 5x3x2x2

The simplest is T=1 (60 subunits)

More complex structures have more subunits: 60 x T

Selection rule: T = h2 + hk + k2 h,k = 0,1,2!

Allowed T-numbers: 1, 3, 4, 7, 9, 12, 13, 16, 19, !

Special T-numbers: 2, 6, ???

h2 + h*k + k2 = T h k T 1 0 1 1 1 3 2 0 4 2 1 7 2 2 12 3 0 9 3 1 13 3 2 19 3 3 27 4 0 16

T = Triangulation

T = 1 T = 3 T = 4

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T=1: Adenovirus 3 Dodecahedral Particle

100• DF DB

Adenovirus 3 Dodecahedron

Hexon

Penton base

Penton base

Fibre

Fuschiotti et al (2006), J Mol Biol 356, 510-510

Dodecahedra in Negative Stain

500•

500•

Structure: Visualization by cryoEM Dodecahedron Movie

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T=4: Hepatitis B Virus

LSBR/NIH: Naiqian Cheng, David Belnap & Alasdair Steven PEL/NIH: Norman Watts, Steve Stahl & Paul Wingfield

!

149 -10 e-Ag

2 183 core-Ag

147 1 Cp147

!

HBV Capsid Movie!

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“Extending Resolution” by Labeling

Conway et al, J Mol Biol 1997 Zlotnick et al, PNAS 1997 Conway et al, PNAS 1998

RNA RNA

Confirmation by Xtallography

Wynne et al, Mol Cell, 1999; PDB 1qgt

Labeling with FabÕ s:"Linear and Conformational Epitopes!

3105! F11A4! 3120! 312! Stain!

Capsids"& Dimers!

Capsids"only!

Peptide!Labels:!

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Fab 312 - model HBV - Fab Complexes

Conway et al, J Virol 2003 Belnap et al, PNAS 2003

HBV - Fab Complexes

3105

3120

312

F11A4

mAb

312 78-83

3105 77-80, 83-84 83-84

1 20 40 60 80 100 120 140 Residues in Epitope

3120 22-22, 25-29, 126-127 20-22, 129-132

F11A4 74-77 78-80, 83-84

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HBV & Fabs: Conclusions

In General: •" 9Å maps, localize specific amino acids by tagging •" Identify conformational epitopes by cryoEM & modeling

•"Occupancy: steric blocking & epitope conformation

•"Diversity: 4 Fabs Ð 4 epitopes

HBV-Specific: •"Distinction between core-antigen and e-antigen

- Accessible surfaces different - Fabs sensitive to small changes in structure (eg, 3120)

•"Structural basis for literature on competition assays - Crowding: almost any pair of Fabs will compete - Competition does not imply overlapping epitopes

149 -10 e-Ag

2 78-83 183 core-Ag

Human papillomavirus T = ??? 5

6 6

5

12 pentavalent, 60 hexavalent sites => 22 + 2*1 + 12 => T=7

U. Tours: Antoine TouzŽ & Pierre Coursaget

72 pentamers: 72 x 5 = 360 subunits, but 360 / 60 => T=6!

T = Triangulation

T = 7d T = 7l

2 1

h=2; k=1 22 + 2*1 + 12

Generalized Capsid Assembly Pathway!

Adapted from Steven, Heymann, Cheng, Trus & Conway (2005) Curr Opin Struct Biol 15, 227-236.

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HK97 Capsid Assembly Pathway!

Adapted from Steven, Heymann, Cheng, Trus & Conway (2005) Curr Opin Struct Biol 15, 227-236.

Phage HK97 capsid structures by cryoEM!

Conway et al, J Mol Biol 1995!

PI! PII! HI! HII!

500• !

200• !

Phage HK97 capsid structures by cryoEM!

Prohead!

200• !

Head!

Conway et al, J Mol Biol 1995!T=7l

Phage HK97 capsid at atomic resolution!

Wikoff et al, Science (2000)!Helgstrand et al, J Mol Biol (2003)!

• isopeptide bond Asn356-Lys169!• auto-catalyzed by Glu363!• branched protein backbone!• catenated rings:"

protein chainmail!

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Phage HK97 procapsids!

100• !

Prohead II!Prohead I!

Conway et al, Science 2001!

Phage HK97 capsid expansion!

Prohead! Head!

Lata et al, Cell 2000!

E X P A N S I O N!

Phage HK97 capsid expansion models! Phage HK97 capsid expansion models!

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Phage HK97 capsid expansion models!Other phage capsids share the HK97 fold!

HK97!gp5!

T4!gp24!

P22!gp5!

Jiang et al, 2003!PNAS!

Fokine et al, 2005!PNAS!

Wikoff et al, 2000!Science!

!29!gp8!

Morais et al, 2005!Molecular Cell!

Other phage capsids share the HK97 fold!

HK97!gp5!

#15!gp7!

T7!gp10!

Agirrezabala et al, 2007!Structure!

Jiang et al, 2008!Nature!

Wikoff et al, 2000!Science!

P22!gp5!

Chen et al, 2011!PNAS!

Herpesvirus capsids also exploit the HK97 fold?!

HK97 gp5

HSV1 VP5

Baker et al, J Virol 2005 - Figure 1!

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The HK97 fold is numerous and diverse ! There are other capsid protein fold families !

Adenovirus! Phage PRD1!

5 5

5

3

2

2 2

T=25

PBCV-1

STIV!STIVSTIV

Capsid protein fold families!

Domain-spanning families of capsid morphology: Ad/PRD1/STIV: pseudo-T=25 or trimeric hexon Reovirus/"6: T=2, asymmetric dimer, multi-layered, dsRNA Herpesvirus/SPO1: T=16, asymmetric triplexes

Emerging lineages of capsid protein fold: Double-barrel-roll: Ad, PRD1, PBCV-1, STIV Asymmetric-dimer: "6, reovirus, rotavirus, L-A HK97: T4 / phi29 / P22 (T7/T5/gifsy-2) Ð HSV-1

Review of 2nd Lecture

• TEM images are projection images and include the entire structure (they are not slices) • Central Section Theorem is the basis for image reconstruction:

Alternatively, back-projection methods in real space • Decay of high frequencies may be compensated by R-weighting (ie, according to radius) or more

sophisticated (but computationally expensive) methods. • Single Particle Analysis Ð many particles oriented randomly • Reconstruction procedure Ð starting model, model-based refinement of orientations • Example 1 (T=1) adenovirus penton base particles • Example 2 (T=3 & T=4) Hepatitis B virus capsid:

9 • resolution structure by cryoEM extend resolution by labeling specific residues and visualizing label position

• Human papillomavirus: morphology to inform on structural details Ð Ò forbiddenÓ T=6 icosahedral geometry

• Phage HK97: capsid dynamics during assembly, including proteolysis and expansion •hybrid cryoEM-Xray crystallography approach for pseudo-atomic resolution