Ishemgulova, A. | Search bioRxiv

bioRxiv2024

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

molecular biology↗

bioRxiv2023

Smart Leginon enables cryoEM screening automation across multiple grids

SummaryCryoEM multi-grid screening is often a tedious process that demands hours of attention. Here, this protocol shows how to set up standard Leginon collection and Smart Leginon Autoscreen to automate this process. This protocol can be applied to the majority of cryoEM holey foil grids. Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to [~]10 minutes with Autoscreen compared to [~]6 hours using previous methods which are hampered by their inability to account for high variability between grids. This protocol and video tutorial first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12 grid automated screening session.

molecular biology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗