Would you like to learn about new cryo-ET methods for your research? Delmic and Alvéole are inviting you to their live mini-symposium where you will discover new research methods from four leading scientists, interact with them, and get to ask questions about their research.
Delmic & Alvéole mini-Symposium: Upcoming trends in cryo-EM
Date : March 22nd, 2023
Place : Virtual conference
Programme of the symposium
Upcoming trends in cryo-EM
WEDNESDAY, MARCH 22nd, 5PM CET (Paris time)
Join this symposium to:
- Learn cryo-ET methods from four leading scientists
- Ask questions to advance your research
- Interact with the cryo-EM community
Alvéole and Delmic both believe that cryo-ET is a powerful technique that will advance our understanding of life at the nanoscale, and we are dedicated to making this technique accessible to everyone.
During this mini-symposium, four cryo-ET scientists will show you their latest cryo-ET results and what methods they used to obtain them.
There will be a Q&A at the end of the webinar so that you can ask all your cryo-ET questions to boost your own research.
A correlative pipeline to quantify complex organellar membrane ultrastructures
Abstract (from our recent article, Quantifying organellar ultrastructure in cryo-electron tomography using a surface morphometrics pipeline, B. Barad et al., JCB, 2023):
Cellular cryo-electron tomography (cryo-ET) enables 3-dimensional reconstructions of organelles in their native cellular environment at subnanometer resolution. However, quantifying ultrastructural features of pleomorphic organelles in three dimensions is challenging, as is defining the significance of observed changes induced by specific cellular perturbations. To address this challenge, we established a semi-automated workflow to segment organellar membranes and reconstruct their underlying surface geometry in cryo-ET. To complement this workflow, we developed an open source suite of ultrastructural quantifications, integrated into a single pipeline called the surface morphometrics toolkit. This toolkit allows detailed mapping of spacing, curvature, and orientation onto reconstructed membrane meshes, highlighting subtle organellar features that are challenging to detect in three dimensions and allowing for statistical comparison across many organelles. To demonstrate the advantages of this approach, we combine cryo-ET with cryo-fluorescence microscopy to correlate bulk mitochondrial network morphology (i.e., elongated versus fragmented) with membrane ultrastructure of individual mitochondria in the presence and absence of endoplasmic reticulum (ER) stress. Using our toolkit, we demonstrate ER stress promotes adaptive remodeling of ultrastructural features of mitochondria including spacing between the inner and outer membranes, local curvature of the inner membrane, and spacing between mitochondrial cristae. We show that differences in membrane ultrastructure correlate to mitochondrial network morphologies, suggesting that these two remodeling events are coupled. Our toolkit offers opportunities for quantifying changes in organellar architecture on a single-cell level using cryo-ET, opening new opportunities to define changes in ultrastructural features induced by diverse types of cellular perturbations.
Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Plasma ion sources can be used for ion beam milling, coupling cryoET lamella fabrication to volumetric workflows utilising both SEM and fluorescence of bulk material (tissue). Their ability to be used at high currents mean large volumes can be milled in relatively short periods of time. I will describe methods aimed at utlising plasma FIB/SEM to streamline lamella fabrication for structure determination that, in combination with serial FIB/SEM and fluorescence, promises a framework for targeting specific features in bulk-frozen samples (>100 µm). I will discuss the promise of these approaches for future developments and how such technologies promise to fit into nascent cryo-CLEM workflows as well as being applied to (human) tissue samples.
Visualizing macromolecular structures in situ by cryo-electron tomography
Structures of purified proteins and protein complexes are now routinely determined to atomic or near-atomic resolutions using single particle cryoEM. Structures of macromolecular assemblies that are intrinsically flexible, dynamic, heterogeneous, and often function in higher-order assemblies, have recently been analyzed to near-atomic resolutions using cryo-electron tomography (cryoET) and subtomogram averaging (STA). The study of native complexes in their cellular context using cryoET STA, coupled with cryoFIB/SEM and correlative and multiscale imaging, opens a new frontier in structural cell biology. Here I present our recent multi-modal, multi-scale imaging of virus infection in cells and in situ structural determination of bacterial enzymes functioning in carbon fixation to demonstrate the power of in situ structural biology using cell lamellae-based cryoET STA.
More details on the talks’ content soon.