Chapter 9
Structural Biology in Situ Using
Cryo-Electron Subtomogram Analysis
Stefan Pfeffer and Friedrich Förster
Abstract Cryo-electron tomography (CET) provides three-dimensional (3D) views
on the structure and organization of a wide range of frozen-hydrated specimens
under near-to-native conditions. In combination with advanced image processing
methods, the locations, orientations and structures of large macromolecular complexes can be mined from tomographic data.
9.1 Introduction
CET and subtomogram analysis allow studying the structure and distribution of
macromolecular complexes without isolation and purification, which is a major
caveat in the structural analysis of large membrane-embedded or transiently associated complexes. CET often complements reductionist structural molecular biology approaches that aim at obtaining high-resolution structures of isolated
components (X-ray crystallography, cryo-EM single particle analysis), putting them
into a cellular context. In this sense CET bridges the traditionally largely separated
disciplines of molecular and cellular structural biology. Here, we provide an
overview of the computational methodology for subtomogram analysis and illustrate the use of these approaches for a set of tomograms depicting endoplasmic
reticulum (ER)-derived vesicles. Using strategies for particle localization, subtomogram alignment and subtomogram classification, a subnanometer resolution
S. Pfeffer Á F. Förster (&)
Department of Molecular Structural Biology, Max-Planck Institute
of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
e-mail: f.g.forster@uu.nl
S. Pfeffer
e-mail: Pfeffer@biochem.mpg.de
F. Förster
Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research,
Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
© Springer International Publishing AG 2018
E. Hanssen (ed.), Cellular Imaging, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-68997-5_9
237
Structural Biology in Situ Using
Cryo-Electron Subtomogram Analysis
Stefan Pfeffer and Friedrich Förster
Abstract Cryo-electron tomography (CET) provides three-dimensional (3D) views
on the structure and organization of a wide range of frozen-hydrated specimens
under near-to-native conditions. In combination with advanced image processing
methods, the locations, orientations and structures of large macromolecular complexes can be mined from tomographic data.
9.1 Introduction
CET and subtomogram analysis allow studying the structure and distribution of
macromolecular complexes without isolation and purification, which is a major
caveat in the structural analysis of large membrane-embedded or transiently associated complexes. CET often complements reductionist structural molecular biology approaches that aim at obtaining high-resolution structures of isolated
components (X-ray crystallography, cryo-EM single particle analysis), putting them
into a cellular context. In this sense CET bridges the traditionally largely separated
disciplines of molecular and cellular structural biology. Here, we provide an
overview of the computational methodology for subtomogram analysis and illustrate the use of these approaches for a set of tomograms depicting endoplasmic
reticulum (ER)-derived vesicles. Using strategies for particle localization, subtomogram alignment and subtomogram classification, a subnanometer resolution
S. Pfeffer Á F. Förster (&)
Department of Molecular Structural Biology, Max-Planck Institute
of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
e-mail: f.g.forster@uu.nl
S. Pfeffer
e-mail: Pfeffer@biochem.mpg.de
F. Förster
Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research,
Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
© Springer International Publishing AG 2018
E. Hanssen (ed.), Cellular Imaging, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-68997-5_9
237