In the process, however, much has been learned about the difficulties to be overcome and insights
obtained into possible routes to do so. In particular:
(i) Three-dimensional (3D) crystals of ternary MP/APol/detergent complexes have been produced, which do diffract X-rays, even though, at this point, to limited resolution (Charvolin
et al. 2014).
(ii) 3D crystals diffracting to very high resolution have been obtained by using APols as a
shuttle to introduce MPs into a lipidic mesophase (Polovinkin et al. 2014; Broecker et al.
2017).
(iii) Satisfying non-ionic APols have been designed, synthesized, and validated (Bazzacco et al.
2012; Sharma et al. 2012; see Chap. 4), although not yet tested for MP crystallization.
These progresses will be reviewed in § 11.3, and challenges and prospects discussed. In § 11.2, a
brief overview will be first provided of the current status of MP crystallization and crystallography,
with the accent placed on developing technologies, particularly inasmuch as they could potentially
benefit from being combined with the use of APols.
11.2 Context: Classical and Less Classical Approaches to Crystallizing
Membrane Proteins
Until the 1980s, MP crystallization, much like MP folding (see Chap. 6), was considered by most
membrane biochemists as an almost hopeless endeavor, and few people were daring enough to
seriously engage in such risky if not doomed attempts. The first well-diffracting crystals were reported
in the early 1980s, by two different groups, for two bacterial MPs, E. coli’s outer membrane porin
OmpF (Garavito and Rosenbusch 1980) and the photosynthetic reaction center from the purple
bacterium Rhodopseudomonas viridis (Michel 1982). Phasing problems delayed the resolution of
the structure of OmpF until the 1990s (Cowan et al. 1992), whereas a 3-Å resolution electron density
map of the reaction center was established in 1985, allowing the construction of the first atomic
molecular model ever to be built of an integral MP in its entirety, i.e. including its transmembrane
(TM) region (Deisenhofer et al. 1984, 1985; Michel et al. 1986; Deisenhofer and Michel 1989).
11.2.1 Factors Contributing to the Steady Growth of the Number
of MP Structures Solved
As every general review on MP crystallization will recall (see e.g. Ostermeier and Michel 1997;
Wiener 2001, 2004; Caffrey 2003, 2009, 2015; Nollert 2004, 2005; Cherezov et al. 2006; Tate 2010;
Cherezov 2011; Ujwal and Bowie 2011; Parker and Newstead 2012; Kang et al. 2013; Ishchenko et al.
2014, 2017; Loll 2014; Moraes et al. 2014), the initial growth in new MP structures was excruciatingly
slow, before progressively picking up steam and embarking onto an exponential course (Fig. 11.1).
The main reason for the initial slow growth was the dearth of MPs that could be obtained in detergent
solution in sufficient amount and under a stable enough form for crystallization trials to be launched.
Most MPs, indeed, are not produced en masse in cells and tissues (Chap. 1), and most of them are
unstable in detergent solutions (Chap. 2). The progressive acceleration in the rate of 3D structures
solved is due to a large number of factors among which are psychological ones (the realization that the
task after all was not as desperate as it had looked earlier, which dramatically increased the number of
people willing to tackle it and the bulk of resources allotted to it), biochemical ones, which we will
498
11 Amphipols and Membrane Protein Crystallization
obtained into possible routes to do so. In particular:
(i) Three-dimensional (3D) crystals of ternary MP/APol/detergent complexes have been produced, which do diffract X-rays, even though, at this point, to limited resolution (Charvolin
et al. 2014).
(ii) 3D crystals diffracting to very high resolution have been obtained by using APols as a
shuttle to introduce MPs into a lipidic mesophase (Polovinkin et al. 2014; Broecker et al.
2017).
(iii) Satisfying non-ionic APols have been designed, synthesized, and validated (Bazzacco et al.
2012; Sharma et al. 2012; see Chap. 4), although not yet tested for MP crystallization.
These progresses will be reviewed in § 11.3, and challenges and prospects discussed. In § 11.2, a
brief overview will be first provided of the current status of MP crystallization and crystallography,
with the accent placed on developing technologies, particularly inasmuch as they could potentially
benefit from being combined with the use of APols.
11.2 Context: Classical and Less Classical Approaches to Crystallizing
Membrane Proteins
Until the 1980s, MP crystallization, much like MP folding (see Chap. 6), was considered by most
membrane biochemists as an almost hopeless endeavor, and few people were daring enough to
seriously engage in such risky if not doomed attempts. The first well-diffracting crystals were reported
in the early 1980s, by two different groups, for two bacterial MPs, E. coli’s outer membrane porin
OmpF (Garavito and Rosenbusch 1980) and the photosynthetic reaction center from the purple
bacterium Rhodopseudomonas viridis (Michel 1982). Phasing problems delayed the resolution of
the structure of OmpF until the 1990s (Cowan et al. 1992), whereas a 3-Å resolution electron density
map of the reaction center was established in 1985, allowing the construction of the first atomic
molecular model ever to be built of an integral MP in its entirety, i.e. including its transmembrane
(TM) region (Deisenhofer et al. 1984, 1985; Michel et al. 1986; Deisenhofer and Michel 1989).
11.2.1 Factors Contributing to the Steady Growth of the Number
of MP Structures Solved
As every general review on MP crystallization will recall (see e.g. Ostermeier and Michel 1997;
Wiener 2001, 2004; Caffrey 2003, 2009, 2015; Nollert 2004, 2005; Cherezov et al. 2006; Tate 2010;
Cherezov 2011; Ujwal and Bowie 2011; Parker and Newstead 2012; Kang et al. 2013; Ishchenko et al.
2014, 2017; Loll 2014; Moraes et al. 2014), the initial growth in new MP structures was excruciatingly
slow, before progressively picking up steam and embarking onto an exponential course (Fig. 11.1).
The main reason for the initial slow growth was the dearth of MPs that could be obtained in detergent
solution in sufficient amount and under a stable enough form for crystallization trials to be launched.
Most MPs, indeed, are not produced en masse in cells and tissues (Chap. 1), and most of them are
unstable in detergent solutions (Chap. 2). The progressive acceleration in the rate of 3D structures
solved is due to a large number of factors among which are psychological ones (the realization that the
task after all was not as desperate as it had looked earlier, which dramatically increased the number of
people willing to tackle it and the bulk of resources allotted to it), biochemical ones, which we will
498
11 Amphipols and Membrane Protein Crystallization