COPII coat, which interacts with the cargo and the membrane, assembles with the
cage-forming outer COPII layer [50].
Other macromolecular membrane protein assemblies that have been studied
using CET and subtomogram analysis are immature retrovirus capsids that are
heterogeneous in size and morphology, favoring CET for their structural analysis.
These highly repetitive structures could be resolved to subnanometer resolution for
three different retroviruses including HIV-1, revealing the tertiary and quaternary
structural interactions mediating virus assembly [51, 52].
Another huge membrane-embedded structure extensively studied using CET and
subtomogram analysis is the bacterial flagellar motor that drives locomotion using
energy stored in a chemical gradient across the bacterial cell membrane.
Subtomogram averages of the flagellar motor from various species revealed its
basic molecular architecture [53], structural diversity [54] and sequential assembly
[55]. Structurally closely related to the flagellar motor are bacterial secretion systems/injectisomes that are used to transfer virulence proteins into eukaryotic host
cells. Studies using CET and subtomogram analysis illuminated the interaction
between host and pathogen cells, providing insights into the extensive conformational changes of secretion systems that drive effector translocation upon host
membrane contact [56, 57]. A size analysis of injectisome basal bodies in single
subtomograms revealed significant variations in length, which is likely required for
many proteins spanning two membranes to accommodate a range of variable
intermembrane distances [58].
Finally, the structure of mitochondrial ATP synthases in the inner mitochondrial
membrane was studied using CET and subtomogram analysis, revealing a conserved dimeric organization in various organisms, mediated by their peripheral
stalks [59, 60]. Analysis of the long-range order of ATP synthase dimers revealed
their distinct arrangement in long double rows along the cristae ridges, likely
inducing their high membrane curvature.
This multitude of successful case studies demonstrates the potential of CET and
subtomogram analysis for structural studies in situ. Recent advances in direct
detector technology, data acquisition strategies and image processing software
promise highly detailed insights into the structure and organization of macromolecules in a cellular context, which will advance our understanding of a cell’s
native molecular landscape significantly, in the future.
9.8 Practical Section: Step-by-Step Guide
for Subtomogram Analysis Using av3 and PyTom
9.8.1 Template Matching and Peak Selection
This section explains how to set up a template matching job and how to select a
number of cross-correlation peaks for further processing in av3/tom and PyTom.
9 Structural Biology in Situ Using Cryo-Electron …
251
cage-forming outer COPII layer [50].
Other macromolecular membrane protein assemblies that have been studied
using CET and subtomogram analysis are immature retrovirus capsids that are
heterogeneous in size and morphology, favoring CET for their structural analysis.
These highly repetitive structures could be resolved to subnanometer resolution for
three different retroviruses including HIV-1, revealing the tertiary and quaternary
structural interactions mediating virus assembly [51, 52].
Another huge membrane-embedded structure extensively studied using CET and
subtomogram analysis is the bacterial flagellar motor that drives locomotion using
energy stored in a chemical gradient across the bacterial cell membrane.
Subtomogram averages of the flagellar motor from various species revealed its
basic molecular architecture [53], structural diversity [54] and sequential assembly
[55]. Structurally closely related to the flagellar motor are bacterial secretion systems/injectisomes that are used to transfer virulence proteins into eukaryotic host
cells. Studies using CET and subtomogram analysis illuminated the interaction
between host and pathogen cells, providing insights into the extensive conformational changes of secretion systems that drive effector translocation upon host
membrane contact [56, 57]. A size analysis of injectisome basal bodies in single
subtomograms revealed significant variations in length, which is likely required for
many proteins spanning two membranes to accommodate a range of variable
intermembrane distances [58].
Finally, the structure of mitochondrial ATP synthases in the inner mitochondrial
membrane was studied using CET and subtomogram analysis, revealing a conserved dimeric organization in various organisms, mediated by their peripheral
stalks [59, 60]. Analysis of the long-range order of ATP synthase dimers revealed
their distinct arrangement in long double rows along the cristae ridges, likely
inducing their high membrane curvature.
This multitude of successful case studies demonstrates the potential of CET and
subtomogram analysis for structural studies in situ. Recent advances in direct
detector technology, data acquisition strategies and image processing software
promise highly detailed insights into the structure and organization of macromolecules in a cellular context, which will advance our understanding of a cell’s
native molecular landscape significantly, in the future.
9.8 Practical Section: Step-by-Step Guide
for Subtomogram Analysis Using av3 and PyTom
9.8.1 Template Matching and Peak Selection
This section explains how to set up a template matching job and how to select a
number of cross-correlation peaks for further processing in av3/tom and PyTom.
9 Structural Biology in Situ Using Cryo-Electron …
251