surfactants tested, only digitonin and A8-35 work well (Yang et al. 2017). The ryanodine receptor
RyR1 has been studied by cryo-EM both in A8-35 (Baker et al. 2015; Study 12.19) and in nanodiscs
(NDs), where its structure was solved to 6.1-Å resolution (Efremov et al. 2015). The structure of
TRPV1 has been solved both in A8-35 (to 3.4 Å) (Cao et al. 2013b; Liao et al. 2013; Study 12.9) and in
NDs (to 2.9–3.4 Å) (Gao et al. 2016). The structure of the respirasome (from different sources) has
been established to 9 Å in A8-35 (Sousa et al. 2016; Study 12.27) and to 5.8–6.7 Å (Letts et al. 2016)
and 5.4 Å (Gu et al. 2016) in digitonin. Similarly, the cryo-EM structures of the Polycystic Kidney
Disease 2 (Pkd2) channel obtained in NDs (to 3-Å resolution) or in A8-35 (to 4 Å) are superimposable
(Shen et al. 2016). Whenever a comparison is possible, the structures solved in A8-35 vs. digitonin or
A8-35 vs. NDs appear extremely similar, suggesting that they all reflect the conformation (or one of the
conformations) the MP or MP complex adopts in the membrane. Which system to choose depends both
on the protein’s preferences and on experimental priorities and convenience.
Subsequent works on APol-trapped TRP channels were carried out by cryo-EM and will be
presented in § 12.3.3.
In 2013, two further studies resorted to trapping with A8-35 followed by NS and yielded 3D
reconstructions consistent with a belt of APol surrounding the TM region. Study 12.10 (Kevany et al.
2013) bore on a heterotetrameric complex of peripherin and ROM1, two retina-specific tetraspanins
that are required for the morphogenesis and maintenance of photoreceptor outer segments. The
complexes were purified from bovine retinas in DDM, supplemented with A8-35, and the detergent
and excess APol removed by SEC. Grids were stained with uranyl acetate and images collected in both
tilted and untilted modes. A 3D reconstruction was calculated to ~18-Å resolution (Fig. 12.14A).
Consideration of the predicted volumes occupied by the TM and extramembrane domains led to their
assignment to the thicker and thinner regions of the model, respectively, the TM region being
surrounded by a 1.5–2-nm-thick belt of APol (dark blue band in Fig. 12.14A; Kevany et al. 2013).
Study 12.11 (Tsybovsky et al. 2013) bore on ABCA4, a member of the ATP-binding cassette
(ABC) family of transporters. ABCA4 is predominantly expressed in the outer segments of rod and
cone photoreceptors and is involved in the clearance from photoreceptors of all-trans-retinal, the
product of light-induced isomerization of 11-cis-retinal. ABCA4 was purified from bovine retinas in
DDM and transferred to A8-35 by SEC in the absence of surfactant, a process that must have removed
both the detergent and free APol. EM grids were negatively stained with uranyl acetate and imaged at
both 0
and 45
angles to the electron beam. An initial 3D model at 38-Å resolution was calculated
from pairs of tilted and untilted images (random conical tilt method) and used as a starting point for
classifying ~25,000 untilted projections, from which a 18-Å-resolution 3D model was calculated
(Fig. 12.14C, D). The intradiscal domain, which is glycosylated, was identified by its binding of
concanavalin. The TM domain was estimated to have a ~2-nm-thick belt of A8-35 bound to its
periphery, in good agreement with the expected dimensions of the TM region (Fig. 12.14E).
Study 12.15 examined the behavior of the trimeric E. coli outer membrane protein OmpF
following transfer from the detergent C 8 -POE to A8-35 using Bio-Beads and elimination of free
APol by SEC, in the presence or absence of lipopolysaccharide (LPS), the main lipidic component of
the outer leaflet of E. coli’s outer membrane (Arunmanee et al. 2014). Within minutes, OmpF trimers
depleted of free APol in the absence of LPS started to organize into long filaments (Fig. 12.15C),
which elongated with time (Fig. 12.15D–F). The thickness of the filaments, ~6 nm, corresponds to the
height of OmpF trimers, normal to the membrane, indicating that they associate side by side
(Fig. 12.15B), as proposed for BR/A8-35 filaments (Gohon et al. 2008; cf. Fig. 12.12 in § 12.3.1).
This suggests that, upon APol depletion, MPs tend to interact hydrophobically via their TM surfaces,
probably releasing some APol in the process. The SEC data show that association occurs in solution,
prior to adsorption onto the EM grids. The filaments interact mostly sideways with the carbon film, that
is, again, via the APol-coated TM surface of OmpF. As observed with the small oligomers formed by
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
557
RyR1 has been studied by cryo-EM both in A8-35 (Baker et al. 2015; Study 12.19) and in nanodiscs
(NDs), where its structure was solved to 6.1-Å resolution (Efremov et al. 2015). The structure of
TRPV1 has been solved both in A8-35 (to 3.4 Å) (Cao et al. 2013b; Liao et al. 2013; Study 12.9) and in
NDs (to 2.9–3.4 Å) (Gao et al. 2016). The structure of the respirasome (from different sources) has
been established to 9 Å in A8-35 (Sousa et al. 2016; Study 12.27) and to 5.8–6.7 Å (Letts et al. 2016)
and 5.4 Å (Gu et al. 2016) in digitonin. Similarly, the cryo-EM structures of the Polycystic Kidney
Disease 2 (Pkd2) channel obtained in NDs (to 3-Å resolution) or in A8-35 (to 4 Å) are superimposable
(Shen et al. 2016). Whenever a comparison is possible, the structures solved in A8-35 vs. digitonin or
A8-35 vs. NDs appear extremely similar, suggesting that they all reflect the conformation (or one of the
conformations) the MP or MP complex adopts in the membrane. Which system to choose depends both
on the protein’s preferences and on experimental priorities and convenience.
Subsequent works on APol-trapped TRP channels were carried out by cryo-EM and will be
presented in § 12.3.3.
In 2013, two further studies resorted to trapping with A8-35 followed by NS and yielded 3D
reconstructions consistent with a belt of APol surrounding the TM region. Study 12.10 (Kevany et al.
2013) bore on a heterotetrameric complex of peripherin and ROM1, two retina-specific tetraspanins
that are required for the morphogenesis and maintenance of photoreceptor outer segments. The
complexes were purified from bovine retinas in DDM, supplemented with A8-35, and the detergent
and excess APol removed by SEC. Grids were stained with uranyl acetate and images collected in both
tilted and untilted modes. A 3D reconstruction was calculated to ~18-Å resolution (Fig. 12.14A).
Consideration of the predicted volumes occupied by the TM and extramembrane domains led to their
assignment to the thicker and thinner regions of the model, respectively, the TM region being
surrounded by a 1.5–2-nm-thick belt of APol (dark blue band in Fig. 12.14A; Kevany et al. 2013).
Study 12.11 (Tsybovsky et al. 2013) bore on ABCA4, a member of the ATP-binding cassette
(ABC) family of transporters. ABCA4 is predominantly expressed in the outer segments of rod and
cone photoreceptors and is involved in the clearance from photoreceptors of all-trans-retinal, the
product of light-induced isomerization of 11-cis-retinal. ABCA4 was purified from bovine retinas in
DDM and transferred to A8-35 by SEC in the absence of surfactant, a process that must have removed
both the detergent and free APol. EM grids were negatively stained with uranyl acetate and imaged at
both 0
and 45
angles to the electron beam. An initial 3D model at 38-Å resolution was calculated
from pairs of tilted and untilted images (random conical tilt method) and used as a starting point for
classifying ~25,000 untilted projections, from which a 18-Å-resolution 3D model was calculated
(Fig. 12.14C, D). The intradiscal domain, which is glycosylated, was identified by its binding of
concanavalin. The TM domain was estimated to have a ~2-nm-thick belt of A8-35 bound to its
periphery, in good agreement with the expected dimensions of the TM region (Fig. 12.14E).
Study 12.15 examined the behavior of the trimeric E. coli outer membrane protein OmpF
following transfer from the detergent C 8 -POE to A8-35 using Bio-Beads and elimination of free
APol by SEC, in the presence or absence of lipopolysaccharide (LPS), the main lipidic component of
the outer leaflet of E. coli’s outer membrane (Arunmanee et al. 2014). Within minutes, OmpF trimers
depleted of free APol in the absence of LPS started to organize into long filaments (Fig. 12.15C),
which elongated with time (Fig. 12.15D–F). The thickness of the filaments, ~6 nm, corresponds to the
height of OmpF trimers, normal to the membrane, indicating that they associate side by side
(Fig. 12.15B), as proposed for BR/A8-35 filaments (Gohon et al. 2008; cf. Fig. 12.12 in § 12.3.1).
This suggests that, upon APol depletion, MPs tend to interact hydrophobically via their TM surfaces,
probably releasing some APol in the process. The SEC data show that association occurs in solution,
prior to adsorption onto the EM grids. The filaments interact mostly sideways with the carbon film, that
is, again, via the APol-coated TM surface of OmpF. As observed with the small oligomers formed by
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
557