other cell types, to detect changes in ambient temperature. TRPV1 is activated by noxious heat and
modulated by inflammatory agents, such as extracellular protons and bioactive lipids, which contribute
to pain hypersensitivity. TRPV1 and other somatosensory TRP channels are considered important
targets for analgesic drugs. A truncated but functional version of the rat receptor was expressed in HEK
cells, solubilized and purified in DDM, and transferred to A8-35 using Bio-Beads, followed by SEC,
which must have eliminated most of the free APol. Freezing in vitreous ice on holey carbon films was
carried out 6 s after blotting. Cryo-EM images were collected using a direct-electron detector and
image processing algorithms to correct motion-induced image blurring and improve signal and
contrast (§ 12.2.3). A 3D reconstruction of TRPV1 in the absence of ligands was built to 3.4-Å
resolution (FSC ¼ 0.143) (Fig. 12.21). This high resolution allowed direct fitting into density maps of
molecular models, particularly in the TM region (Fig. 12.22) (Liao et al. 2013). In a companion paper,
Fig. 12.19 3D map of the respirasome (mitochondrial supercomplex B) and fitted X-ray structures.
(A) Cryo-EM 3D map as seen from two opposite sides (left), from the matrix (top right), and from the
intermembrane space (lower right). The amphipol belt is shown in brown. The circle marks the gap
between Complex I and Complex III. (B) X-ray structures of component complexes (blue, Complex I; red,
Complex III; green, Complex IV) and cytochrome c (black) fitted to the 3D cryo-EM map. (C) Docked
X-ray structures without map. (D) Enlarged view with docked X-ray structures. The mobile, electronshuffling domains of the two Rieske proteins are shown in orange. The arrow points to the gap between
the two matrix domains of Complex III, which was used for positioning the X-ray structure. MA matrix,
M membrane, IM intermembrane space. Scale bars: 10 nm (From Althoff et al. 2011, # 2011 European
Molecular Biology Organization).
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
565
modulated by inflammatory agents, such as extracellular protons and bioactive lipids, which contribute
to pain hypersensitivity. TRPV1 and other somatosensory TRP channels are considered important
targets for analgesic drugs. A truncated but functional version of the rat receptor was expressed in HEK
cells, solubilized and purified in DDM, and transferred to A8-35 using Bio-Beads, followed by SEC,
which must have eliminated most of the free APol. Freezing in vitreous ice on holey carbon films was
carried out 6 s after blotting. Cryo-EM images were collected using a direct-electron detector and
image processing algorithms to correct motion-induced image blurring and improve signal and
contrast (§ 12.2.3). A 3D reconstruction of TRPV1 in the absence of ligands was built to 3.4-Å
resolution (FSC ¼ 0.143) (Fig. 12.21). This high resolution allowed direct fitting into density maps of
molecular models, particularly in the TM region (Fig. 12.22) (Liao et al. 2013). In a companion paper,
Fig. 12.19 3D map of the respirasome (mitochondrial supercomplex B) and fitted X-ray structures.
(A) Cryo-EM 3D map as seen from two opposite sides (left), from the matrix (top right), and from the
intermembrane space (lower right). The amphipol belt is shown in brown. The circle marks the gap
between Complex I and Complex III. (B) X-ray structures of component complexes (blue, Complex I; red,
Complex III; green, Complex IV) and cytochrome c (black) fitted to the 3D cryo-EM map. (C) Docked
X-ray structures without map. (D) Enlarged view with docked X-ray structures. The mobile, electronshuffling domains of the two Rieske proteins are shown in orange. The arrow points to the gap between
the two matrix domains of Complex III, which was used for positioning the X-ray structure. MA matrix,
M membrane, IM intermembrane space. Scale bars: 10 nm (From Althoff et al. 2011, # 2011 European
Molecular Biology Organization).
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
565