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Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegansworm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure. A technique to ‘lift out’ samples of interest from high-pressure-frozen specimens expands applications of cryo-electron tomography to multicellular organisms and tissue.

Botulinum neurotoxin serotype A1 (BoNT/A1), a licensed drug widely used for medical and cosmetic applications, exerts its action by invading motoneurons. Here we report a 2.0-Å-resolution crystal structure of the BoNT/A1 receptor-binding domain in complex with its neuronal receptor, glycosylated human SV2C. We found that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—which is conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and for its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an antibotulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications, thereby achieving highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.

Abstract Shallow wakes that occur in a wide range of natural flows tend to generate instabilities that develop into large, 2D coherent structures (2DCS). We present the results of an experimental study to stabilize shallow wakes by local, enhanced bottom roughness. Two successful stabilization strategies are compared to a base case of an unsteady bubble wake. First, localized bed roughness is placed in the lateral shear layers near the shoulders of the cylinder. Second, a local roughness element is placed at the end of the recirculation bubble, in the downstream region where large-scale vortices would normally shed. Dye visualization is used to assess the qualitative behavior of the wake, and two-component laser Doppler velocimetry (LDV) measurements are made to measure the Reynolds stress distributions and time-averaged velocity profiles. In both stabilization cases, a minimum patch size of the enhanced roughness elements is required for stabilization, which depends on the momentum thickness of the shear layers and the locations of enhanced Reynolds shear stresses. The main effect of the wake stabilization is a reduction in momentum exchange with the ambient flow due to damping of the large 2DCS. This reduction in eddy diffusivity results in a narrower wake and a slower decay of the centerline velocity deficit with downstream distance compared to the base case of an unsteady bubble wake.