1. N EAR -A TOMIC R ESOLUTION A CHIEVED U SING C RYO -EM Lindsey Organtini 8-16-13 Structure Work Group

2. V IRUS 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

3. V IRUSES IN C RYO EM 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

4. F UN F ACT As of 2010, ~20% of all entries have achieved resolutions better than 10Å!

5. T HE I MPORTANCE OF R ESOLUTION 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

6. W HY RESOLUTION MATTERS … Not near atomic, but improved resolution can show make a big difference in interpretation!

7. N- TERMINI OF EV71 NOT RESOLVED IN CRYSTAL STRUCTURE

8. W E ’ VE SEEN WHAT HIGH RESOLUTION CAN ACHIEVE, BUT WHAT ABOUT NEAR - ATOMIC RESOLUTION ?

9. R IBSOME DETAILS WITH INCREASING RESOLUTION

10. A T 3.8Å, SEE HELICES AND SHEETS IN R OTAVIRUS VP6

11. N EW D ISCOVERIES IN Ε 15 P HAGE Previous reconstruction 9.5Å -> Added 20,000 more particles to achieve 4.5Å gp7 gp10

12. L ITTLE SEQUENCE BUT HIGH STRUCTURAL SIMILIARITIES CryoEM shows subtle differences between the three structures

13. 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 A DVANCES IN A DENOVIRUS

14. M INOR PROTEINS IN A DENOVIRUS 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

15. P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES Provirion 3.8Å with 23,4000 particles Virus 4.0 Å with18,3000 particles Virion is 100 Å wider and more angular than provirion Hexamers skewed in provirion which become more symmetric in virion

16. P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES Cyan = procapsid Magenta = virion

17. P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES Provirion 3.8Å with 23,4000 particles Virus 4.0 Å with18,3000 particles Virion is 100 Å wider and more angular than provirion Hexamers skewed in provirion which become more symmetric in virion

18. P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES Procapsid Virion Between capsomeres Between asymmetric units

19. S O 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...

21. T HERE IS STILL A PLACE FOR X - RAY CRYSTALLOGRAPHY !

22. CryoEM + X-ray Combination of both for pseudo atomic resolution Pseudo Atomic Modeling Example (Virus + FAb) Fragment of Ab

24. I MAGINE THE INFORMATION WE COULD ACHIEVE BY COMBINING NEAR - ATOMIC RESOLUTION CRYO EM AND X - RAY DATA !