(images of RyR1/A8-35 complexes are reported to have an ~2 to 6% contrast at 5–10-Å resolution as
compared to 0–2% in the presence of CHAPS, using a traditional CCD detector). However, the RyR1/
A8-35 particles tend to adopt a uniform orientation, with their long axis normal to the plane of the film
(Fig. 12.26B). Supplementing the preparation with a submicellar concentration of octylglucoside
(OG) (~10% of the CMC) results in a random distribution of orientations (Fig. 12.26C). This is one
of the few studies where hypotheses about the reasons for improved cryo-EM data collected with
APol-trapped vs. detergent-solubilized MPs are critically discussed.
As already mentioned, cryo-EM studies of APol-stabilized MPs are being published at a rapid
rate. Studies published or located after this chapter was written and that could not be included in
Table 12.1 or discussed here include Wei et al. 2016; Xu et al. 2016; Arnaud et al. 2017; Bausewein
et al. 2017; Beckham et al. 2017; Chiu et al. 2017; Ekiert et al. 2017; Fitzpatrick et al. 2017; Li et al.
2017a; Punjani et al. 2017; Schoebel et al. 2017; Terahara et al. 2017; and Zhou et al. 2017.
12.3.4 Conclusions and Perspectives
From the overview of published works presented above, it appears that APols have integrated the field
of EM and look likely to continue to be helpful in the preparation of improved samples and in their
imaging. A discussion of their plusses and minuses and of useful steps that could be taken can be split
into two main issues: (i) the solution properties of APol-trapped vs. detergent-solubilized samples and
(ii) issues related to imaging.
12.3.4.1 Solution Properties of the Samples
The effects of APols on the MPs they trap have been discussed in Chap. 5. They relate essentially to
three distinct mechanisms, whose impact on EM studies, however, is difficult to fathom from the
published literature:
(i) APol-trapped MPs are generally more stable than their detergent-solubilized counterparts
(Chap. 5, § 5.5). For MP oligomers, this means, for instance, that the risk of monomerization
or the loss of subunits is reduced. By the same token, a monomeric MP stands more chance of
being observed in its native conformation. This effect will reduce the heterogeneity of the
preparations and the risk that the painfully reconstructed 3D model does not correspond to a
native state.
Not all MPs are stabilized by APols. As discussed in more detail in Chap. 5, when stability is
an issue, steps that can be taken include comparing the effects of different APols (there are
suggestions that the least charged APols are more stabilizing – see e.g. Picard et al. 2006;
Dahmane et al. 2009; Bazzacco et al. 2012 – but this may not be universal, cf. Huynh et al.
2014b), supplementing the preparation with a small amount of lipids (see e.g. Dahmane et al.
2013), and/or, perhaps, playing on the ionic strength: whereas this point has not yet been
experimentally examined, it seems likely that electrostatic repulsion between MP-adsorbed
APols plays a role in destabilization and that raising the ionic strength could mitigate this effect.
Needless to say, working in the presence of high-affinity ligands is a stabilizing factor.
(ii) There are indications that APols may affect the dynamics of MPs, through the “Gulliver effect”
discussed in Chap. 5, namely a (hypothetical) increase in the free energy barrier between various
conformational states, which would slow down or block transitions. There are some indications,
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
573
compared to 0–2% in the presence of CHAPS, using a traditional CCD detector). However, the RyR1/
A8-35 particles tend to adopt a uniform orientation, with their long axis normal to the plane of the film
(Fig. 12.26B). Supplementing the preparation with a submicellar concentration of octylglucoside
(OG) (~10% of the CMC) results in a random distribution of orientations (Fig. 12.26C). This is one
of the few studies where hypotheses about the reasons for improved cryo-EM data collected with
APol-trapped vs. detergent-solubilized MPs are critically discussed.
As already mentioned, cryo-EM studies of APol-stabilized MPs are being published at a rapid
rate. Studies published or located after this chapter was written and that could not be included in
Table 12.1 or discussed here include Wei et al. 2016; Xu et al. 2016; Arnaud et al. 2017; Bausewein
et al. 2017; Beckham et al. 2017; Chiu et al. 2017; Ekiert et al. 2017; Fitzpatrick et al. 2017; Li et al.
2017a; Punjani et al. 2017; Schoebel et al. 2017; Terahara et al. 2017; and Zhou et al. 2017.
12.3.4 Conclusions and Perspectives
From the overview of published works presented above, it appears that APols have integrated the field
of EM and look likely to continue to be helpful in the preparation of improved samples and in their
imaging. A discussion of their plusses and minuses and of useful steps that could be taken can be split
into two main issues: (i) the solution properties of APol-trapped vs. detergent-solubilized samples and
(ii) issues related to imaging.
12.3.4.1 Solution Properties of the Samples
The effects of APols on the MPs they trap have been discussed in Chap. 5. They relate essentially to
three distinct mechanisms, whose impact on EM studies, however, is difficult to fathom from the
published literature:
(i) APol-trapped MPs are generally more stable than their detergent-solubilized counterparts
(Chap. 5, § 5.5). For MP oligomers, this means, for instance, that the risk of monomerization
or the loss of subunits is reduced. By the same token, a monomeric MP stands more chance of
being observed in its native conformation. This effect will reduce the heterogeneity of the
preparations and the risk that the painfully reconstructed 3D model does not correspond to a
native state.
Not all MPs are stabilized by APols. As discussed in more detail in Chap. 5, when stability is
an issue, steps that can be taken include comparing the effects of different APols (there are
suggestions that the least charged APols are more stabilizing – see e.g. Picard et al. 2006;
Dahmane et al. 2009; Bazzacco et al. 2012 – but this may not be universal, cf. Huynh et al.
2014b), supplementing the preparation with a small amount of lipids (see e.g. Dahmane et al.
2013), and/or, perhaps, playing on the ionic strength: whereas this point has not yet been
experimentally examined, it seems likely that electrostatic repulsion between MP-adsorbed
APols plays a role in destabilization and that raising the ionic strength could mitigate this effect.
Needless to say, working in the presence of high-affinity ligands is a stabilizing factor.
(ii) There are indications that APols may affect the dynamics of MPs, through the “Gulliver effect”
discussed in Chap. 5, namely a (hypothetical) increase in the free energy barrier between various
conformational states, which would slow down or block transitions. There are some indications,
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
573