NEAR-ATOMIC RESOLUTION ACHIEVED USING CRYO-EMLindsey Organtini8-16-13Structure Work Group

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

FUN FACT

As of 2010, ~20% of all entries have achieved resolutions better than 10Å!

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!

N-TERMINI OF EV71 NOT RESOLVED IN CRYSTAL STRUCTURE

WE’VE SEEN WHAT HIGH RESOLUTION CAN ACHIEVE, BUT WHAT ABOUT NEAR-ATOMIC RESOLUTION?

RIBSOME DETAILS WITH INCREASING RESOLUTION

AT 3.8Å, SEE HELICES AND SHEETS IN ROTAVIRUS VP6

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

Cyan = procapsidMagenta = 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 . . .

THERE IS STILL A PLACE FOR X-RAY CRYSTALLOGRAPHY!

CryoEM + X-ray Combination of both for pseudo atomic resolution

Pseudo Atomic Modeling Example (Virus + FAb)

Fragment of Ab

IMAGINE THE INFORMATION WE COULD ACHIEVE BY COMBINING NEAR-ATOMIC RESOLUTION CRYOEM AND X-RAY DATA!