NEAR-ATOMIC RESOLUTION ACHIEVED USING CRYO-EM Lindsey Organtini 8-16-13 Structure 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 gp7 gp10 Previous reconstruction 9.5Å -> Added 20,000 more particles to achieve 4.5Å LITTLE SEQUENCE BUT HIGH STRUCTURAL SIMILIARITIES CryoEM shows subtle differences between the three structures ADVANCES IN ADENOVIRUS 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 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 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 P22 CAPTURED IN MULTIPLE CONFORMATIONAL STATES Cyan = procapsid Magenta = virion 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 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! Pseudo Atomic Modeling Example (Virus + FAb) Fragment of Ab CryoEM + X-ray Combination of both for pseudo atomic resolution IMAGINE THE INFORMATION WE COULD ACHIEVE BY COMBINING NEAR-ATOMIC RESOLUTION CRYOEM AND XRAY DATA!