Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages

Abstract

Advances in electron cryo-tomography have provided a new opportunity to visualize the internal 3D structures of a bacterium. An electron microscope equipped with Zernike phase contrast optics produces images with dramatically increased contrast compared to images obtained by conventional electron microscopy. Here we describe a protocol to apply Zernike phase plate technology for acquiring electron tomographic tilt series of cyanophage-infected cyanobacterial cells embedded in ice, without staining or chemical fixation. We detail the procedures for aligning and assessing phase plates for data collection, and methods to obtain 3D structures of cyanophage assembly intermediates in the host, by subtomogram alignment, classification and averaging. Acquiring three to four tomographic tilt series takes approximately 12 h on a JEM2200FS electron microscope. We expect this time requirement to decrease substantially as the technique matures. Time required for annotation and subtomogram averaging varies widely depending on the project goals and data volume.

Membrane protein structures without crystals, by single particle electron cryomicroscopy

“…Recent analysis of the ATPase dimers in the inner mitochondrial membrane, membrane associated mitochondrial ribosomes and the nicotinic acetylcholine receptor with rapsyn on the post-synaptic membranes of the electric organ of Torpedo highlight such possibilities for membrane proteins [ 88 , 89 , 90 ]. Two recent advances namely the gold supported grids that minimize some beam-induced movement [ 91• , 92• ] and the phase plate [ 93 , 94 ] have great potential in acquiring better images by tomography and increased resolution by sub-tomogram averaging.…”

Setting the dynein motor in motion: New insights from electron tomography

“…2A), but are free of the dehydration and staining artifacts introduced by epoxy-embedding methodologies (141). By combining cryoelectron tomography data collection schemes with phase plate technology, which dramatically increases the phase contrast within EM images, researchers can image these thin lamellae to produce unprecedented views of the subcellular organization of thousands of macromolecules pristinely preserved with minimal perturbation to their native in situ structure (142)(143)(144)(145). Furthermore, technological improvements to CLEM approaches offer the exciting promise of improving both the spatial and temporal correlation between complementary imaging modalities (reviewed in Ref.…”

The integrative role of cryo electron microscopy in molecular and cellular structural biology

“…Two important areas to address these are (i) the instrumentation and (ii) software developments. Instrumentation (i) comprises for example high‐resolution electron microscopes, direct electron detector cameras with higher DQE at high frequencies, energy filters to remove inelastically scattered electrons and reduce the background in the images, correction of the spherical aberration (Cs) to improve the optical system in the column of the microscope, microelectron diffraction to determine the structure from small 3D crystals (Nederlof et al., ; Sawaya et al., ; Shi et al., ), spraying of small amounts on cryo‐EM grids (Chen et al., ; Razinkov et al., ), cryo transfer between FIB and electron microscope for cryo‐ET (Schaffer et al., ) and contrast‐increasing phase plates placed in the back‐focal plane of the microscope (Danev et al., ; Dai et al., ; Frindt et al., ; Walter et al., ; Chua et al., ; Danev et al., ; Glaeser, ; Rhinow ; Khoshouei et al., ); phase plates could facilitate structure determination of relatively small complexes (Khoshouei et al., ), which were difficult to address previously and usually limited to lower resolution (Baird et al., ; Orlov et al., ; Maletta et al., ) even though DDDs have helped a lot moving forward (Merk et al., ), suggesting that synergies will appear for example between phase plates, energy filters and high‐sensitivity cameras to enable high‐contrast high‐resolution image acquisition (a feature that usually contradicts itself considering the requirement of defocussing during data collection to get some reasonable amount of image contrast required for image processing). Further software developments (ii) will be required for automatic data acquisition for massive data collection for single particle cryo‐EM and cryo‐ET ( e.g .…”