Category: Past Talks

Alberto Bartesaghi

Associate Professor of Computer Science, Biochemistry and Electrical and Computer Engineering, Duke University

CryoET – high-speed or high-resolution? BISECT tackles both

Date and time: Dec 2nd, 2020 Wednesday, at 8am PT / 11am ET / 4pm GMT / 5pm CET / Dec 3rd midnight China

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Tomographic reconstruction of cryo-preserved specimens followed by extraction and averaging of sub-volumes has been successfully used to determine the structure of macromolecules in their native environment.
Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. Delays introduced during mechanical navigation of the specimen and stage tilting, however, significantly slow down data collection thus limiting its practical value. Here, I present BISECT (beam image-shift electron cryo-tomography), a new protocol to accelerate tilt-series acquisition without sacrificing resolution. I also describe improvements to our constrained single particle tomography (CSPT) framework, leading to higher resolution reconstructions determined by sub-volume averaging.
For validation, we collected tilt-series from a low molecular weight target (~300kDa) using BISECT and processed them using CSPT to obtain a 3.6 Å resolution map where density for side chains is clearly resolved. These advances bring cryo-ET a step closer to becoming a high-throughput tool for in-situ structure determination at near-atomic resolution.

Bridget Carragher
Director, Simons Electron Microscopy Center,
New York Structural Biology Center, NY, NY, USA

CryoEM: Challenges and Opportunities

Date and time: Nov 4th, 2020 Wednesday, at 8am PT / 11am ET / 4pm BST / 5pm CET / Nov 5th midnight China

Bridget will talk about challenges and opportunities in cryoEM; then, we will invite participants to add their perspective. What are the things that existing algorithms and software cannot do currently? What would make experimental work better? What do we expect or hope to achieve in the coming years?

Edward H Egelman
Professor, Biochemistry and Molecular Genetics, University of Virginia

Cryo-EM and Helical Polymers: A Natural Affinity

Date and time: 9/23/2020 Wednesday, at 8am PT / 11am ET/ 4pm BST / 5pm CEST / 11pm China

Link: please join our mailing list at https://mailman.yale.edu/mailman/listinfo/ow_cryoem

It was over fifty years ago that the first 3D reconstruction from electron microscopy was published (DeRosier and Klug, 1968), and this happened to be a helical phage tail. It was no accident that this was a helical specimen, and not just because helical polymers are so abundant in biology. From one point of view helical objects are the simplest to reconstruct: a single image of a helical filament may provide all of the information needed for a 3D reconstruction. Fourier-Bessel methods of reconstruction dominated the field for the next 30 years, but now real-space approaches have become the standard. With the advent of direct-electron detectors, reaching a near-atomic resolution for helical polymers has become the standard, rather than the exception. However, problems in determining the correct helical symmetry can persist even when one reaches a resolution of ~ 5 Å. I will discuss how the best approach to determining helical symmetry involves an averaged power spectrum from the images, and explain why the power spectrum of an averaged image (such as using Class2D) is not the same as the averaged power spectrum. In addition, the highly anisotropic environment present in thin films prior to vitrification, the compressional forces associated with these thin films, and fluid flow with associated shear, can all impact the structure of the filaments being examined. I will discuss these problems and potential solutions while giving an overview of some of our own efforts involving everything from viruses infecting hosts living in nearly boiling acid to microbial nanowires, with stops along the way at bacterial and archaeal flagellar filaments and bacterial and archaeal pili.

Images courtesy of Agnieszka Kawska.