Elden R. Strahm Emeritus Professor of Structural Virology
Ghost protocol: Seeing the invisible with cryoEM variance maps.
All imaging techniques based on multiple copies of the object imaged (e.g. X-ray crystallography and cryoEM reconstructions) depend on closely similar appearances from one observation of the object to the next. Unlike crystallography, CryoEM places no constraint on the object and at the moment of freezing a biological specimen can assume a range of spatial occupancies that depend on the intrinsic dynamic restraints of the specimen. A novel analytical approach to cryoEM reconstructions will be presented that characterizes the continuous 3 dimensional heterogeneity of an ensemble of particles resulting in variance maps that reveal dynamic features that are not obvious in the cryoEM density. Three examples will be discussed where variance maps revealed intrinsic, localized particle heterogeneity associated with chemical mechanisms of virus maturation. (Wang, Q., Matsui, T., Domitrovic, T., Zheng, Y., Doerschuk, P. C., and Johnson, J. E. 2013. Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps. J Struct Biol 181:195-206).
Florida State University
Automated Fiducial-less Tilt-Series Alignment with Protomo.
For more than a decade, tilt-series collection using gold fiducial beads has allowed for structural determination of in situ cellular structures as well as heterogeneous macromolecular structures. However, not all specimen are amenable to the use of fiducials. The emergence of direct electron detectors has enabled the collection of low dose, high contrast images, opening the door for reliable correlation-based tilt-series alignment. I will present a set of automated, correlation-based tilt-series alignment, CTF correction, and reconstruction workflows from within Appion with the goal of making the production of high quality tomograms accessible to all users.