Use of Cryo-EM to Study the Structure of Chemoreceptor Arrays In Vivo
Cryo-electron microscopy (cryo-EM) allows the imaging of intact macromolecular complexes in the context of whole cells. The biological samples for cryo-EM are kept in a near-native state by flash freezing, without the need for any additional sample preparation or fixation steps. Since transmission electron microscopy only generates 2D projections of the samples, the specimen has to be tilted in order to recover its 3D structural information. This is done by collecting images of the sample with various tilt angles in respect to the electron beam. The acquired tilt series can then be computationally back-projected. This technique is called electron cryotomography (ECT), and has been instrumental in unraveling the architecture of chemoreceptor arrays. Here we describe the method of visualizing in vivo bacterial chemoreceptor arrays in three main steps: immobilization of bacterial cells on EM grids by plunge-freezing; 2D image acquisition in tilt series; and 3D tomogram reconstruction.
KeywordsCryo-EM Cryotomography Plunge freezing Chemoreceptor arrays
We thank Dr. Christoph Diebolder from NeCEN for helpful comments to improve the manuscript.
- 14.Frank J (ed) (2006) Electron tomography. methods for three-dimensional visualization of structures in the cell, 2nd edn. Springer, New York, NYGoogle Scholar
- 33.Chen S, McDowall A, Dobro MJ, Briegel A, Ladinsky M et al (2010) Electron cryotomography of bacterial cells. J Vis Exp 39. pii:1943Google Scholar