title: Cryo-Electron Tomography and Subtomogram Averaging.
abstract: Cryo-electron tomography (cryo-ET) allows 3D volumes to be reconstructed from a set of 2D projection images of a tilted biological sample. It allows densities to be resolved in 3D that would otherwise overlap in 2D projection images. Cryo-ET can be applied to resolve structural features in complex native environments, such as within the cell. Analogous to single-particle reconstruction in cryo-electron microscopy, structures present in multiple copies within tomograms can be extracted, aligned, and averaged, thus increasing the signal-to-noise ratio and resolution. This reconstruction approach, termed subtomogram averaging, can be used to determine protein structures in situ. It can also be applied to facilitate more conventional 2D image analysis approaches. In this chapter, we provide an introduction to cryo-ET and subtomogram averaging. We describe the overall workflow, including tomographic data collection, preprocessing, tomogram reconstruction, subtomogram alignment and averaging, classification, and postprocessing. We consider theoretical issues and practical considerations for each step in the workflow, along with descriptions of recent methodological advances and remaining limitations.
authors: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors: W
authors: W
authors: Wan
authors: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. Electronic address: john.briggs@embl.de.
authors: J A G
authors: JA
authors: Briggs
mesh_terms: D000465:Algorithms; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation / Q000379:methods*; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods / Q000706:statistics & numerical data*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D011506:Proteins / Q000648:ultrastructure; D059629:Signal-To-Noise Ratio; D012984:Software; D057188:Workflow
chemical_list: D011506:Proteins
keywords: Cryo-electron microscopy; Cryo-electron tomography; Image processing; In situ structural biology; Subtomogram averaging