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Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50 years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50 nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The structure determination of integral membrane protein (IMP) in lipid environment is particularly challenging. Among emerging methods for exchanging detergent required for IMP purification by original compounds, the use of lipid nanodisc preserves a lipid environment. Compared with the classical method of proteoliposome formation, the nanodisc technology provides a better control of IMP molecules inserted in lipid membrane, therefore giving access to structural methodologies developed for soluble proteins. Here, we present the reconstitution of OprM membrane protein into nanodisc associated with a step of size-exclusion chromatography, an approach applicable to prepare IMPs for subsequent visualization by single-particle electron microscopy.