Box 9.2 X-ray and Neutron Scattering
In broad terms, X-ray and neutron scattering, and which information can be gathered from them, are
not conceptually difficult: in the same way as photons reflected by your environment, focused by
your eye lens, detected by your retina, and analyzed by your brain will tell you whether you are
facing a charging elephant or a pack of hamsters (and, hopefully, trigger the appropriate response),
the photons or neutrons scattered by an ensemble of biological objects of nanometric dimensions
contain information about their size, shape, composition, internal organization, interactions, and
dynamics. The Devil, however, is in the details: the physics of radiation scattering is quite intricate,
and its mathematics is discouraging to the normally constituted biochemist. The success and
reliability of “small-angle” scattering experiments (in short, SAXS for X-rays and SANS for
neutrons) therefore rely on the combined expertise involved in carefully preparing appropriate
samples and rigorously collecting, correcting, and analyzing scattering data, which most often
depends on setting up appropriate collaborations. It is essential, however, that the biochemist know
what is doable and what to pay attention to and why, and the physicist or biophysicist have some
notions of what dirty tricks biochemical samples may have up their sleeves. The present box has no
other objective than to offer this basic background. For a more detailed and rigorous treatment, the
reader is referred to such textbooks as Cantor and Schimmel (1980), Svergun et al. (2013), and
Zaccai et al. (2017).
Here are a few key points:
• Origin of the contrast and how to modify it. Neutrons interact with nuclei and X-rays
with electrons. This has various consequences that make the two approaches complementary. The contrast between particles and solvent depends, for X-rays, on their
respective electron density and, for neutrons, on the properties of their nuclei, measured
by a parameter, ρ, known as the “scattering length density.” Elements with a high atomic
number carry more electrons and scatter X-rays more strongly than lighter elements.
Such is not the case with neutrons, the interaction with which depends on nuclear
properties that do not vary monotonically. X-rays are insensitive to isotopic labeling,
whereas in SANS, the contrast between various components of a particle and between
them and the solvent can be modulated by it. In particular,
1
H nuclei scatter neutrons in
opposition of phase with all other common nuclei, including
2
H, in effect subtracting
their contribution from that of most other atoms (which is why the scattering length
density of
1
H 2 O is negative; see Fig. 9.16A). This has two extremely useful
consequences:
– First, by preparing buffers in appropriately chosen H 2 O/D 2 O mixtures, it is
possible to “contrast-match” one or another component of a complex particle,
e.g. to make “invisible,” at least at low resolution, the detergent or the APol
that keeps a MP soluble. Figure 9.16A illustrates how the contrast between the
solvent and various molecules changes as a function of the percentage of D 2 O
in the solvent (for an overview, see Jacrot 1976). Proteins, for instance, have a
point of zero contrast (“contrast-match point,” CMP) of ~40% D 2 O (pink
line), whereas lipids, which are richer in
1
H, need only ~10% D 2 O (purple
line). A8-35 is contrast-matched at ~23.5% D 2 O (Gohon et al. 2004)
(Fig. 9.16B). Note that the hydrophobic effect is stronger in D 2 O, which
tends to favor aggregation. It is therefore advisable, when transferring a
sample to D 2 O or H 2 O/D 2 O mixtures, to check on the absence of
perturbations, e.g. by SAXS or AUC.
– Second, by deuterating selectively one of the components of a particle, its
contrast with H 2 O is enormously increased and its CMP displaced to much
higher D 2 O concentrations (see deuterated protein and DNA in Fig. 9.16A).
Two deuterated versions of A8-35 are available: one whose side chains only are
deuterated, with a CMP of ~85% D 2 O (Gohon et al. 2004; Fig. 9.16B), and the
other perdeuterated, with a CMP above 100% D 2 O (Giusti et al. 2014). Working
in D 2 O-rich buffers that contrast-match deuterated APols has two great
advantages. First, it increases the contrast between the MP and the solvent;
second, it diminishes considerably the background noise, a large fraction
428
9 Solution Studies of Membrane Protein/Amphipol Complexes
In broad terms, X-ray and neutron scattering, and which information can be gathered from them, are
not conceptually difficult: in the same way as photons reflected by your environment, focused by
your eye lens, detected by your retina, and analyzed by your brain will tell you whether you are
facing a charging elephant or a pack of hamsters (and, hopefully, trigger the appropriate response),
the photons or neutrons scattered by an ensemble of biological objects of nanometric dimensions
contain information about their size, shape, composition, internal organization, interactions, and
dynamics. The Devil, however, is in the details: the physics of radiation scattering is quite intricate,
and its mathematics is discouraging to the normally constituted biochemist. The success and
reliability of “small-angle” scattering experiments (in short, SAXS for X-rays and SANS for
neutrons) therefore rely on the combined expertise involved in carefully preparing appropriate
samples and rigorously collecting, correcting, and analyzing scattering data, which most often
depends on setting up appropriate collaborations. It is essential, however, that the biochemist know
what is doable and what to pay attention to and why, and the physicist or biophysicist have some
notions of what dirty tricks biochemical samples may have up their sleeves. The present box has no
other objective than to offer this basic background. For a more detailed and rigorous treatment, the
reader is referred to such textbooks as Cantor and Schimmel (1980), Svergun et al. (2013), and
Zaccai et al. (2017).
Here are a few key points:
• Origin of the contrast and how to modify it. Neutrons interact with nuclei and X-rays
with electrons. This has various consequences that make the two approaches complementary. The contrast between particles and solvent depends, for X-rays, on their
respective electron density and, for neutrons, on the properties of their nuclei, measured
by a parameter, ρ, known as the “scattering length density.” Elements with a high atomic
number carry more electrons and scatter X-rays more strongly than lighter elements.
Such is not the case with neutrons, the interaction with which depends on nuclear
properties that do not vary monotonically. X-rays are insensitive to isotopic labeling,
whereas in SANS, the contrast between various components of a particle and between
them and the solvent can be modulated by it. In particular,
1
H nuclei scatter neutrons in
opposition of phase with all other common nuclei, including
2
H, in effect subtracting
their contribution from that of most other atoms (which is why the scattering length
density of
1
H 2 O is negative; see Fig. 9.16A). This has two extremely useful
consequences:
– First, by preparing buffers in appropriately chosen H 2 O/D 2 O mixtures, it is
possible to “contrast-match” one or another component of a complex particle,
e.g. to make “invisible,” at least at low resolution, the detergent or the APol
that keeps a MP soluble. Figure 9.16A illustrates how the contrast between the
solvent and various molecules changes as a function of the percentage of D 2 O
in the solvent (for an overview, see Jacrot 1976). Proteins, for instance, have a
point of zero contrast (“contrast-match point,” CMP) of ~40% D 2 O (pink
line), whereas lipids, which are richer in
1
H, need only ~10% D 2 O (purple
line). A8-35 is contrast-matched at ~23.5% D 2 O (Gohon et al. 2004)
(Fig. 9.16B). Note that the hydrophobic effect is stronger in D 2 O, which
tends to favor aggregation. It is therefore advisable, when transferring a
sample to D 2 O or H 2 O/D 2 O mixtures, to check on the absence of
perturbations, e.g. by SAXS or AUC.
– Second, by deuterating selectively one of the components of a particle, its
contrast with H 2 O is enormously increased and its CMP displaced to much
higher D 2 O concentrations (see deuterated protein and DNA in Fig. 9.16A).
Two deuterated versions of A8-35 are available: one whose side chains only are
deuterated, with a CMP of ~85% D 2 O (Gohon et al. 2004; Fig. 9.16B), and the
other perdeuterated, with a CMP above 100% D 2 O (Giusti et al. 2014). Working
in D 2 O-rich buffers that contrast-match deuterated APols has two great
advantages. First, it increases the contrast between the MP and the solvent;
second, it diminishes considerably the background noise, a large fraction
428
9 Solution Studies of Membrane Protein/Amphipol Complexes