The Use of Amphipols for Electron
Microscopy
12
Summary
Electron microscopy is, to date, the field of structural biology to which amphipols have
contributed most, to the point that testing amphipols has become a common practice
at the onset of any single-particle high-resolution study of a membrane protein
(MP) by electron cryomicroscopy (cryo-EM). Yet, few methodological studies have
been published. In many cases, it has been shown that the use of amphipols facilitates
cryo-EM studies as compared to detergent solutions and results in better data, but the
origins of this improvement seem to be multiple and have not all been sorted out.
Mechanisms that have some degree of credibility include (i) biochemical stabilization
and, at least in some cases, reduction of the variability of the images of the target MP,
presumably due to limitation of its dynamics; (ii) improved contrast, due to the absence
or near-absence of free surfactant in the solution; and (iii) better spread of the particles
in the water film stretched over the holes in the supporting carbon film, probably
related to surface tension issues.
12.1 Introduction
The application of amphipols (APols) to electron microscopy (EM) developed relatively late. The first
structural biology study of an APol-trapped membrane protein (MP) was actually an EM one (Tribet
et al. 1998; Study 12.1 in Table 12.1 below). However, it did not resort to standard transmission EM
(TEM), but to scanning TEM (STEM). STEM of freeze-dried, unstained specimens does not aim at
obtaining high-resolution images, but at mapping the distribution of mass over the supporting carbon
film. By integration, the mass of each particle can be determined (for a recent review, see Vahedi-Faridi
et al. 2013). The aim of this study was to determine, using preparations of dimers and monomers of the
cytochrome b 6 f complex, whether the mass distribution of APol-trapped particles reflected that
characterized in detergent solution by analytical ultracentrifugation (AUC) (it did; see § 12.3.1). A
series of TEM studies of negatively stained, APol-trapped F 1 F O ATP synthase were published in
1998–2000 (Wilkens and Capaldi 1998a, b; Wilkens 2000; Wilkens et al. 2000; Study 12.2). They
long remained unnoticed by the APol community because APols were mentioned neither in the title
nor in the keywords or abstracts of the papers, the samples being simply described as “detergent-free.”
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_12
533
Microscopy
12
Summary
Electron microscopy is, to date, the field of structural biology to which amphipols have
contributed most, to the point that testing amphipols has become a common practice
at the onset of any single-particle high-resolution study of a membrane protein
(MP) by electron cryomicroscopy (cryo-EM). Yet, few methodological studies have
been published. In many cases, it has been shown that the use of amphipols facilitates
cryo-EM studies as compared to detergent solutions and results in better data, but the
origins of this improvement seem to be multiple and have not all been sorted out.
Mechanisms that have some degree of credibility include (i) biochemical stabilization
and, at least in some cases, reduction of the variability of the images of the target MP,
presumably due to limitation of its dynamics; (ii) improved contrast, due to the absence
or near-absence of free surfactant in the solution; and (iii) better spread of the particles
in the water film stretched over the holes in the supporting carbon film, probably
related to surface tension issues.
12.1 Introduction
The application of amphipols (APols) to electron microscopy (EM) developed relatively late. The first
structural biology study of an APol-trapped membrane protein (MP) was actually an EM one (Tribet
et al. 1998; Study 12.1 in Table 12.1 below). However, it did not resort to standard transmission EM
(TEM), but to scanning TEM (STEM). STEM of freeze-dried, unstained specimens does not aim at
obtaining high-resolution images, but at mapping the distribution of mass over the supporting carbon
film. By integration, the mass of each particle can be determined (for a recent review, see Vahedi-Faridi
et al. 2013). The aim of this study was to determine, using preparations of dimers and monomers of the
cytochrome b 6 f complex, whether the mass distribution of APol-trapped particles reflected that
characterized in detergent solution by analytical ultracentrifugation (AUC) (it did; see § 12.3.1). A
series of TEM studies of negatively stained, APol-trapped F 1 F O ATP synthase were published in
1998–2000 (Wilkens and Capaldi 1998a, b; Wilkens 2000; Wilkens et al. 2000; Study 12.2). They
long remained unnoticed by the APol community because APols were mentioned neither in the title
nor in the keywords or abstracts of the papers, the samples being simply described as “detergent-free.”
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_12
533