n ear -a tomic r esolution a chieved u sing c ryo -em lindsey organtini 8-16-13 structure work group
TRANSCRIPT
VIRUS ASSEMBLY
Structure is a key element in understanding viral assembly
X-Ray crystallography can resolve atomic structural information, so why not use it? Stringent requirements for crystallization not suitable for all
functional states Can suspend particles in specific state using vitreous ice
VIRUSES IN CRYOEM
Particularly suited for cryoEM due to their high symmetry, molecular mass, stability, and solubility in buffers
Have been used since the inception of cryoEM De Rosier and Klug used T4 bacteriophage tails in their 1970
paper
THE IMPORTANCE OF RESOLUTION
Improving resolution means more structural features are discernible Low resolutions (20-10 Å)= general shape, capsomere
morphology High Resolution (9-6 Å) = individual subunit boundaries,
secondary structure elements (α helices, β sheets) Near Atomic Resolution (<4.5 Å) = Pitch of helices,
separation of β strands, some side chains of a.a. Able to determine features unable to be crystallized Can use both in conjunction in order to learn more
WHY RESOLUTION MATTERS…
Not near atomic, but improved resolution can show make a big difference in interpretation!
NEW DISCOVERIES IN Ε15 PHAGE
Previous reconstruction 9.5Å -> Added 20,000 more particles to achieve 4.5Å
gp7
gp10
LITTLE SEQUENCE BUT HIGH STRUCTURAL SIMILIARITIES
CryoEM shows subtle
differences between the
three structures
31,815 particles used to achieve 3.6Å of 2 major proteins (hexon-trimers and penton base)
Reveals N terminal arm not resolvable in X-ray
Similar to arm of rotavirus, which was also revealed by cryoEM and unresolvable in X-ray
ADVANCES IN ADENOVIRUS
MINOR PROTEINS IN ADENOVIRUS
Used to attach major proteins onto lattice
3 proteins resolved high enough to model
Able to detect side chains
X-ray could only resolve 2 proteins partially
P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES
Provirion3.8Å with 23,4000
particles
Virus4.0 Å with18,3000
particles
Virion is 100 Å wider and more angular than provirionHexamers skewed in provirion which become more symmetric in virion
P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES
Provirion3.8Å with 23,4000
particles
Virus4.0 Å with18,3000
particles
Virion is 100 Å wider and more angular than provirionHexamers skewed in provirion which become more symmetric in virion
P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES
Procapsid
Virion
Between capsomeres
Between asymmetric units
SO HOW DO YOU ACHIEVE NEAR-ATOMIC RESOLUTION?
Use many, many particles (10x what is normally used) Automated data collection Will need the computer resources
High quality images No lens aberration or drift CCDs cause information lose
Improved defocus measurements and avoiding alignment error . . .
CryoEM + X-ray Combination of both for pseudo atomic resolution
Pseudo Atomic Modeling Example (Virus + FAb)
Fragment of Ab