U. Pitt. MBSB-1 Fall-2014 Methods, Samples & Symmetry...
Transcript of U. Pitt. MBSB-1 Fall-2014 Methods, Samples & Symmetry...
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Molecular Electron Microscopy Cryo-Electron Microscopy
James Conway BST-3, Room 2047 [email protected] 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¡ !
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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
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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:
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• 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