activity and chosen for EM studies. Samples negatively stained with uranyl acetate showed a high
density of homogeneous, well-preserved particles, of which ~5600 were selected for initial singleparticle processing. Class averages showed the characteristic triangular and F-shaped views of
supercomplex B that had been previously observed in negative stain (Schäfer et al. 2006). Other
class averages, which had not been reported before, were round or oval. As a control, ~4700 digitoninsolubilized supercomplexes were examined after NS. They were less uniform but also revealed a
number of round or oval class averages, indicating that the rounded shapes were not due to the APol.
As no significant differences were observed between supercomplexes trapped by A8-35 or solubilized
in digitonin, the A8-35-trapped material was used for the 3D reconstruction, considering the generally
higher stability of membrane proteins in APols, the better separation on density gradients, and the
absence of detergent, which facilitates cryo-EM grid preparation.
For cryo-EM, samples were vitrified in a thin layer of buffer on continuous carbon support films.
This is not often done in single-particle cryo-EM, because the ice thickness is more difficult to control
than on holey carbon films, and the support film adds background noise. However, the preferential
orientations of the complexes on the support film help in particle selection and classification, and it
becomes easier to use the random conical tilt method of 3D reconstruction (Radermacher et al. 1987).
This approach has the advantage that angular relationships between the particle images are known,
providing important constraints in the initial rounds of reconstruction, which facilitated sorting the
particles into subpopulations. Two images of each area were recorded at tilt angles of 0
and À45
(Fig. 12.18A, B). Five characteristic views out of the final set of 150 class averages from untilted
images are shown in Fig. 12.18C. A 3D map was generated and refined by projection matching to a
resolution of 19 Å as determined at FSC ¼ 0.143. Projections of the final 3D map are compared with
the corresponding class averages in Fig. 12.18D.
The X-ray structures of the three individual respiratory chain complexes were docked manually
into the 19-Å map (Fig. 12.19). The fit of the three complexes left unoccupied an irregular, belt-shaped
region of density around the perimeter of the complex. This band (brownish in Fig. 12.19A) correlates
closely with the hydrophobic TM surface of Complexes I, III, and IV and accordingly was assigned to
A8-35, providing the first direct view of a MP-associated APol belt. The belt is on average ~2 nm thick,
and ~4 nm wide, comparable in width to the hydrophobic region of a lipid bilayer (Lewis and
Engelman 1983). Its thickness is consistent with the estimates of 1.5–2 nm that had been obtained
from SANS and other indirect techniques (Zoonens et al. 2005; Gohon et al. 2008; see Chap. 5, § 5.3.2)
and with that observed in subsequent EM studies (see e.g. Figs. 12.14, 12.23, and 12.25). The belt
showed a number of ~3 nm protrusions, spaced about 6–10 nm apart, suggesting an irregular
distribution along the edge of the membrane region. These bumps could correspond either to
deformations of the belt, or, perhaps, to the attachment of A8-35 particles (which are, however,
somewhat larger: ~6.3 nm in diameter; Gohon et al. 2006; Chap. 4, § 4.3.1.2.2). Whatever their
origin, comparison of supercomplex 3D reconstructions generated from two equal halves of the data
set indicates that the bumps are a distinct map feature, implying that they attach to, or form at, the same
positions in each supercomplex.
The cryo-EM structure of supercomplex B made it possible to map the arrangement of the three
complexes relative to each other and to delineate possible paths for the transfer of ubiquinol/ubiquinone between Complex I and the bc 1 complex, as well as that of cytochrome c between the bc 1 and
cytochrome c oxidase, and to determine the distances between enzymatic sites. The resolution was
later pushed to 9 Å, providing further functional insights into the transfer of electrons from Complex III
to Complex IV (Study 12.27; Sousa et al. 2016).
The publication of the respirasome structure seems to have finally attracted the attention of
microscopists to the possible interest of using APols for cryo-EM studies; it was followed by a flurry of
other works, 22 at the date of this writing (mid-2017), plus one study, also listed in Table 12.1,
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
563
density of homogeneous, well-preserved particles, of which ~5600 were selected for initial singleparticle processing. Class averages showed the characteristic triangular and F-shaped views of
supercomplex B that had been previously observed in negative stain (Schäfer et al. 2006). Other
class averages, which had not been reported before, were round or oval. As a control, ~4700 digitoninsolubilized supercomplexes were examined after NS. They were less uniform but also revealed a
number of round or oval class averages, indicating that the rounded shapes were not due to the APol.
As no significant differences were observed between supercomplexes trapped by A8-35 or solubilized
in digitonin, the A8-35-trapped material was used for the 3D reconstruction, considering the generally
higher stability of membrane proteins in APols, the better separation on density gradients, and the
absence of detergent, which facilitates cryo-EM grid preparation.
For cryo-EM, samples were vitrified in a thin layer of buffer on continuous carbon support films.
This is not often done in single-particle cryo-EM, because the ice thickness is more difficult to control
than on holey carbon films, and the support film adds background noise. However, the preferential
orientations of the complexes on the support film help in particle selection and classification, and it
becomes easier to use the random conical tilt method of 3D reconstruction (Radermacher et al. 1987).
This approach has the advantage that angular relationships between the particle images are known,
providing important constraints in the initial rounds of reconstruction, which facilitated sorting the
particles into subpopulations. Two images of each area were recorded at tilt angles of 0
and À45
(Fig. 12.18A, B). Five characteristic views out of the final set of 150 class averages from untilted
images are shown in Fig. 12.18C. A 3D map was generated and refined by projection matching to a
resolution of 19 Å as determined at FSC ¼ 0.143. Projections of the final 3D map are compared with
the corresponding class averages in Fig. 12.18D.
The X-ray structures of the three individual respiratory chain complexes were docked manually
into the 19-Å map (Fig. 12.19). The fit of the three complexes left unoccupied an irregular, belt-shaped
region of density around the perimeter of the complex. This band (brownish in Fig. 12.19A) correlates
closely with the hydrophobic TM surface of Complexes I, III, and IV and accordingly was assigned to
A8-35, providing the first direct view of a MP-associated APol belt. The belt is on average ~2 nm thick,
and ~4 nm wide, comparable in width to the hydrophobic region of a lipid bilayer (Lewis and
Engelman 1983). Its thickness is consistent with the estimates of 1.5–2 nm that had been obtained
from SANS and other indirect techniques (Zoonens et al. 2005; Gohon et al. 2008; see Chap. 5, § 5.3.2)
and with that observed in subsequent EM studies (see e.g. Figs. 12.14, 12.23, and 12.25). The belt
showed a number of ~3 nm protrusions, spaced about 6–10 nm apart, suggesting an irregular
distribution along the edge of the membrane region. These bumps could correspond either to
deformations of the belt, or, perhaps, to the attachment of A8-35 particles (which are, however,
somewhat larger: ~6.3 nm in diameter; Gohon et al. 2006; Chap. 4, § 4.3.1.2.2). Whatever their
origin, comparison of supercomplex 3D reconstructions generated from two equal halves of the data
set indicates that the bumps are a distinct map feature, implying that they attach to, or form at, the same
positions in each supercomplex.
The cryo-EM structure of supercomplex B made it possible to map the arrangement of the three
complexes relative to each other and to delineate possible paths for the transfer of ubiquinol/ubiquinone between Complex I and the bc 1 complex, as well as that of cytochrome c between the bc 1 and
cytochrome c oxidase, and to determine the distances between enzymatic sites. The resolution was
later pushed to 9 Å, providing further functional insights into the transfer of electrons from Complex III
to Complex IV (Study 12.27; Sousa et al. 2016).
The publication of the respirasome structure seems to have finally attracted the attention of
microscopists to the possible interest of using APols for cryo-EM studies; it was followed by a flurry of
other works, 22 at the date of this writing (mid-2017), plus one study, also listed in Table 12.1,
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
563