12.3.4.3 Perspectives, Suggestions, and Speculations
In concluding this chapter, I would like to point to some areas where there seems to be room for
progress.
(i) A couple of practical suggestions
• Documentation of experimental conditions and methodological studies. It would be desirable
that the exact conditions under which the samples are prepared be more precisely documented. As discussed in the preceding section, experimental details such as the extent of
removal of free APol or the time that elapses between blotting the cryo-EM grid and freezing
it may well play important roles in the distribution and orientation of the particles in the
aqueous film. Now that the usefulness of APols for EM studies is well-established, it would
be helpful to carry out more systematic studies of the importance of the various parameters
that may affect this behavior, including ionic strength, the nature of the APol, and the
presence of traces of detergent. As APols with various chemical structures, charge densities,
etc. have been developed and validated for handling MPs (see Chaps. 4 and 5), it would be
desirable that their usefulness for EM studies be investigated. Note that APols are miscible
with detergents (Zoonens et al. 2007; Tribet et al. 2009; see Chap. 4) and, very likely, at least
for some of them, with one another, so that many parameters, such as charge density, could be
varied in a continuous manner and their effect investigated without having to multiply
syntheses.
• A related question is that of the commercial availability of a range of APols. We have
repeatedly noted, in our survey of the literature, that a surfactant that gives excellent results
with a MP complex may be unsatisfactory with another. It would be highly desirable that, at
the onset of an EM study, a range of different APols be tested. This is more easily said than
done, however, because it is not straightforward to convince a private company to market a
variety of molecules, each of which calls for upscaling and optimizing a synthesis protocol,
when the volumes of sales will, according to all probability, remain small. Perhaps a consortium of potential users should be set up with the aim of contracting for the synthesis
of medium- to large-scale batches of a selection of molecules, which could then be made
available to the community as a whole (see the general “Conclusions and Perspectives”
(§ 12.3.4) at the end of this chapter).
(ii) Some more or less wild speculations
Many opportunities offered by APols have not been exploited for EM yet. Let me list a few
of them:
• Visualizing the APol belt. This could be useful, particularly in low- or medium-resolution
studies, when the position of the TM region in the averaged images can be ambiguous. One
can think of several approaches. Several tagged APols are by now available, carrying a biotin
(Charvolin et al. 2009; Ferrandez et al. 2014), an oligonucleotide (Le Bon et al. 2014a), or a
polyhistidine tag (Giusti et al. 2015) (reviewed in Le Bon et al. 2014b, and in Chap. 4, § 4.4).
They can be used to tie gold nanobeads or monomeric avidin to the TM region of the protein
under study. It is not impossible that the oligonucleotides, which are 6–7 kDa in mass, could
be directly detected or hybridized to a larger marker. The position of the tags will necessarily
vary from one particle to the next, as there is no reason that they should adopt specific
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
577
In concluding this chapter, I would like to point to some areas where there seems to be room for
progress.
(i) A couple of practical suggestions
• Documentation of experimental conditions and methodological studies. It would be desirable
that the exact conditions under which the samples are prepared be more precisely documented. As discussed in the preceding section, experimental details such as the extent of
removal of free APol or the time that elapses between blotting the cryo-EM grid and freezing
it may well play important roles in the distribution and orientation of the particles in the
aqueous film. Now that the usefulness of APols for EM studies is well-established, it would
be helpful to carry out more systematic studies of the importance of the various parameters
that may affect this behavior, including ionic strength, the nature of the APol, and the
presence of traces of detergent. As APols with various chemical structures, charge densities,
etc. have been developed and validated for handling MPs (see Chaps. 4 and 5), it would be
desirable that their usefulness for EM studies be investigated. Note that APols are miscible
with detergents (Zoonens et al. 2007; Tribet et al. 2009; see Chap. 4) and, very likely, at least
for some of them, with one another, so that many parameters, such as charge density, could be
varied in a continuous manner and their effect investigated without having to multiply
syntheses.
• A related question is that of the commercial availability of a range of APols. We have
repeatedly noted, in our survey of the literature, that a surfactant that gives excellent results
with a MP complex may be unsatisfactory with another. It would be highly desirable that, at
the onset of an EM study, a range of different APols be tested. This is more easily said than
done, however, because it is not straightforward to convince a private company to market a
variety of molecules, each of which calls for upscaling and optimizing a synthesis protocol,
when the volumes of sales will, according to all probability, remain small. Perhaps a consortium of potential users should be set up with the aim of contracting for the synthesis
of medium- to large-scale batches of a selection of molecules, which could then be made
available to the community as a whole (see the general “Conclusions and Perspectives”
(§ 12.3.4) at the end of this chapter).
(ii) Some more or less wild speculations
Many opportunities offered by APols have not been exploited for EM yet. Let me list a few
of them:
• Visualizing the APol belt. This could be useful, particularly in low- or medium-resolution
studies, when the position of the TM region in the averaged images can be ambiguous. One
can think of several approaches. Several tagged APols are by now available, carrying a biotin
(Charvolin et al. 2009; Ferrandez et al. 2014), an oligonucleotide (Le Bon et al. 2014a), or a
polyhistidine tag (Giusti et al. 2015) (reviewed in Le Bon et al. 2014b, and in Chap. 4, § 4.4).
They can be used to tie gold nanobeads or monomeric avidin to the TM region of the protein
under study. It is not impossible that the oligonucleotides, which are 6–7 kDa in mass, could
be directly detected or hybridized to a larger marker. The position of the tags will necessarily
vary from one particle to the next, as there is no reason that they should adopt specific
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies
577