The first study by TEM of vitrified specimens observed at cryogenic temperatures (electron
cryomicroscopy, cryo-EM) of an APol-trapped MP, that of mitochondrial Complex I, was started in
1997 but appeared in print only in 2007 (Flötenmeyer et al. 2007; Study 12.3). It established the
feasibility of the approach, but, for reasons that had nothing to do with the use of APols (see below,
§ 12.3.1), the three-dimensional (3D) reconstruction was limited to a very low resolution (58 Å), which
may have, mistakenly, dissuaded potential users from giving APols a try. A study of negatively stained
bacteriorhodopsin (BR) appeared in 2008 as part of a larger-scale characterization of BR/APol
complexes (Gohon et al. 2008; Study 12.4). EM studies however really picked up steam only after
the publication of the respirasome structure by cryo-EM in 2011 (Althoff et al. 2011; Study 12.5;
§ 12.3.2). They have been making up for lost time ever since, with, by mid-2017, >40 studies
published, representing as many proteins or protein complexes and >50 publications (see below,
Table 12.1, and additional publications listed in §§ 12.3.2 and 12.3.3).
In the following, we will first examine the context, namely which EM approaches are applied to
MPs and, in particular, how the samples are prepared (§ 12.2). By a fortunate coincidence, at the time
when electron microscopists were finally discovering the potential interest of using APols for studying
MPs, great technological progress was being accomplished as regards the collection, correction, and
analysis of cryo-EM images, which have revolutionized the field (see below, § 12.2.3). APols bring
their little stone to this progress by improving specimen preparation and visualization (§ 12.3).
12.2 Context: Electron Microscopy Studies of Membrane Proteins
12.2.1 The Various Approaches to Studying Membrane Proteins by Electron
Microscopy
Electron microscopy can be applied to small MPs (e.g. BR, 27 kDa), to huge complexes (e.g. the
respirasome, 1.7 MDa), to subcellular structures, such as mitochondria or synaptic terminals, or even to
whole cells. The nature of the samples and techniques varies widely depending on the target. For small
objects (typically 150 kDa), one will principally resort to two-dimensional (2D) crystals, in the form
either of sheets or tubes, which will be studied by electron crystallography and by Fourier transform
analysis of EM images (for recent reviews, see Raunser and Walz 2009; Ubarretxena-Belandia and
Stokes 2010; Fujiyoshi 2011; Wisedchaisri et al. 2011; Kühlbrandt 2013; Stahlberg et al. 2015). Above
~150 kDa, one can either resort to 2D crystals, if the protein or complex accepts to crystallize, or
reconstruct 3D images by combining tens or hundreds of thousands of projection images of single
particles (see below, § 12.2.2). For whole cells or large subcellular structures, one will use electron
tomography, in which a 3D reconstruction of a single object is calculated from images obtained at
various tilts with respect to the electron beam (for reviews, see e.g. Leis et al. 2009; Lučič et al. 2013;
Villa et al. 2013).
One of the major difficulties of EM of biological samples is to control radiation-induced damage
(for a detailed discussion and comparison with X-ray crystallography, see Henderson 1995). This
limits the doses that can be applied and, therefore, the amount of information afforded by each image.
There are two main ways around this problem. One is to freeze the sample at cryogenic temperatures,
typically those of liquid nitrogen or helium. As this is not sufficient to permit collecting complete data
on a single molecule, data must be averaged from many thousands of individual molecules, each of
which has interacted with a limited number of electrons. This is achieved either by crystallographic
analysis of 2D crystals, in which the molecules are oriented with respect to each other and significant
data can be separated from noise by Fourier analysis, or by aligning images of single particles and
averaging them – the aligning procedure being a complex one. As pointed out early on by Richard
534
12 The Use of Amphipols for Electron Microscopy
cryomicroscopy, cryo-EM) of an APol-trapped MP, that of mitochondrial Complex I, was started in
1997 but appeared in print only in 2007 (Flötenmeyer et al. 2007; Study 12.3). It established the
feasibility of the approach, but, for reasons that had nothing to do with the use of APols (see below,
§ 12.3.1), the three-dimensional (3D) reconstruction was limited to a very low resolution (58 Å), which
may have, mistakenly, dissuaded potential users from giving APols a try. A study of negatively stained
bacteriorhodopsin (BR) appeared in 2008 as part of a larger-scale characterization of BR/APol
complexes (Gohon et al. 2008; Study 12.4). EM studies however really picked up steam only after
the publication of the respirasome structure by cryo-EM in 2011 (Althoff et al. 2011; Study 12.5;
§ 12.3.2). They have been making up for lost time ever since, with, by mid-2017, >40 studies
published, representing as many proteins or protein complexes and >50 publications (see below,
Table 12.1, and additional publications listed in §§ 12.3.2 and 12.3.3).
In the following, we will first examine the context, namely which EM approaches are applied to
MPs and, in particular, how the samples are prepared (§ 12.2). By a fortunate coincidence, at the time
when electron microscopists were finally discovering the potential interest of using APols for studying
MPs, great technological progress was being accomplished as regards the collection, correction, and
analysis of cryo-EM images, which have revolutionized the field (see below, § 12.2.3). APols bring
their little stone to this progress by improving specimen preparation and visualization (§ 12.3).
12.2 Context: Electron Microscopy Studies of Membrane Proteins
12.2.1 The Various Approaches to Studying Membrane Proteins by Electron
Microscopy
Electron microscopy can be applied to small MPs (e.g. BR, 27 kDa), to huge complexes (e.g. the
respirasome, 1.7 MDa), to subcellular structures, such as mitochondria or synaptic terminals, or even to
whole cells. The nature of the samples and techniques varies widely depending on the target. For small
objects (typically 150 kDa), one will principally resort to two-dimensional (2D) crystals, in the form
either of sheets or tubes, which will be studied by electron crystallography and by Fourier transform
analysis of EM images (for recent reviews, see Raunser and Walz 2009; Ubarretxena-Belandia and
Stokes 2010; Fujiyoshi 2011; Wisedchaisri et al. 2011; Kühlbrandt 2013; Stahlberg et al. 2015). Above
~150 kDa, one can either resort to 2D crystals, if the protein or complex accepts to crystallize, or
reconstruct 3D images by combining tens or hundreds of thousands of projection images of single
particles (see below, § 12.2.2). For whole cells or large subcellular structures, one will use electron
tomography, in which a 3D reconstruction of a single object is calculated from images obtained at
various tilts with respect to the electron beam (for reviews, see e.g. Leis et al. 2009; Lučič et al. 2013;
Villa et al. 2013).
One of the major difficulties of EM of biological samples is to control radiation-induced damage
(for a detailed discussion and comparison with X-ray crystallography, see Henderson 1995). This
limits the doses that can be applied and, therefore, the amount of information afforded by each image.
There are two main ways around this problem. One is to freeze the sample at cryogenic temperatures,
typically those of liquid nitrogen or helium. As this is not sufficient to permit collecting complete data
on a single molecule, data must be averaged from many thousands of individual molecules, each of
which has interacted with a limited number of electrons. This is achieved either by crystallographic
analysis of 2D crystals, in which the molecules are oriented with respect to each other and significant
data can be separated from noise by Fourier analysis, or by aligning images of single particles and
averaging them – the aligning procedure being a complex one. As pointed out early on by Richard
534
12 The Use of Amphipols for Electron Microscopy