Protocols in Current Issue
Protocols in Past Issues
0 Q&A 964 Views Feb 20, 2024

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples.

Key features

• Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET).

• Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms.

• The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Graphical overview

0 Q&A 815 Views Jul 20, 2023

Hepatitis B virus (HBV) infection is a global public health concern. During chronic infection, the HBV small-surface antigen is expressed in large excess as non-infectious spherical subviral particles (SVPs), which possess strong immunogenicity. To date, attempts at understanding the structure of HBV spherical SVP have been restricted to 12–30 Å with contradictory conclusions regarding its architecture. We have used cryo-electron microscopy (cryo-EM) and 3D image reconstruction to solve the HBV spherical SVP to 6.3 Å. Here, we present an extended protocol on combining AlphaFold2 prediction with a moderate-resolution cryo-EM density map to build a reliable 3D model. This protocol utilizes multiple software packages that are routinely used in the cryo-EM community. The workflow includes 3D model prediction, model evaluation, rigid-body fitting, flexible fitting, real-space refinement, model validation, and model adjustment. Finally, the described protocol can also be applied to high-resolution cryo-EM datasets (2–4 Å).

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