The use of neutrons in biological research
introduction
crystallography
fibre diffraction
small-angle neutron scattering and associated modelling
specular reflection and off-specular diffraction
lamellar diffraction
incoherent neutron scattering
small molecule structure determination
instrument access and funding

Small-angle neutron scattering can be used to define the location and shape of individual components of large macromolecular complexes. Here, the top row shows two views of the Escherichia coli 70S ribosome electron cryomicroscopy 3-dimensional reconstruction (orange pixels) and the dummy atom model built based on the EM reconstruction to obtain a search volume for neutron data modeling (purple circles 30S subunit dummy atoms; aquamarine circles 50S subunit dummy atoms). The bottom row shows the same two views of the 70S ribosome with different components of the structure found within the search volume coloured distinctly (green 30S subunit proteins; pink 30S subunit rRNA; mauve 50S subunit proteins; ochre 50S subunit rRNA. See elsewhere in this site; figure adapted from images kindly provided by Dmitri Svergun.

What's the use?

The interaction of neutrons with matter is far less strong than that of X-rays or electrons. It is thus possible to collect more data much faster using X-rays or electrons than using neutrons. Nevertheless, the wavelength of radiation which is determinant of the resolution in experimental data is similar for all three approaches. For an electron microscope operating at an accelerating voltage of 100 kV the wavelength of the incident beam is ~0.05 Å. In X-ray diffraction experiments the wavelength will typically be of the order of 0.5-2 Å. In neutron experiments the incident beam can have wavelengths in the range 0.2-10 Å. Electron and X-ray sources frequently lead to specimen damage; but cooling to liquid nitrogen temperatures (or, in the case of electron microscopy liquid helium temperatures) and care in the way in which data are collected minimize this restriction which is in any case offset by the speed of data collection and their coherence. Furthermore, the pre-eminence of X-ray diffraction over electrons or neutrons as an approach in structural biology is firmly established due to the colossal endeavour which would be required to realize the theoretical resolving capability of an electron beam (which is comparable to the resolution practically possible with X-rays). For single-particle approaches to structure determination it has been estimated that this achievement would require the analysis of millions of images whose inherently low contrast would render the definition of their mutual relationship and therefore relationship to the 3-dimensional structure of the specimen with sufficient accuracy immensely difficult. For 2-dimensional electron crystallography, the resolution of structures solved is usually highly anisotropic. Nevertheless, as demonstrated by the fact that reconstructions of virus structures and the structure of the ribosome in the resolution range 7-10 Å have now been obtained, electron cryo-microscopy (cryo-EM) is a powerful and informative complementary technique to X-ray diffraction.

What, then, is the advantage in using neutrons? The main further advantages offered by neutrons over X-rays or electrons are as follows:

- the anomalous signals obtained from hydrogen and its heavier isotope deuterium, leading to

- the ability to label components of a structure without damaging its native conformation by simple substitution of ²H for ¹H, or alternatively

- the ability differentially to visualize components of a macromolecular complex by modulation of the ²H₂O content of the buffer (contrast variation; also possible with X-rays by the inclusion of additional electron dense components in the buffer such as caesium chloride or sucrose). These factors mean

- neutrons are particularly aptly applied to questions concerning the hydration or ionization state of samples, or the relative arrangment of different components of large macromolecular complexes (ribosomes, protein/membrane systems) which may not be amenable to other techniques or where a neutron experiment can provide important accessory information aiding data interpretation and

- a further use of neutrons is the determination of the magnetic structures of materials; particularly applied to superconductors, this has also proved important in work on drug structures and a variant (spin-dependent neutron scattering) has been applied to research on the ribosome.

Further reading:
Henderson, R. Quart. Rev. Biophys. 28, 171-193.
Grimes, J.M., Fuller, S.D. and Stuart, D.I. Acta Crystallogr. D55, 1742-1749.

Technical basis of neutron scattering techniques

Neutron scattering and X-ray scattering are analogous in that they both reveal information concerning the structure and dynamics of the system under investigation. However, whereas X-rays are photons that are scattered by electrons, neutrons, which are scattered from atomic nuclei, are fundamental particles requiring a nuclear reactor or spallation source for their generation. X-rays, on the other hand, may be generated easily in the laboratory as well as using a synchrotron. Furthermore, X-ray scattering varies in a simple way with atomic mass whereas neutron scattering amplitudes depend in a complex manner on the nuclear mass, spin and energy levels of the scattering atom. Neutrons may be scattered coherently or incoherently by nuclei, providing information on the structure of a system or its dynamics respectively. Coherent scattering arises from correlations between the neutrons scattered from nuclei within a system. Incoherent scattering is spatially isotropic and arises from correlations between the same nuclei at time zero and a later time t. If the energy of a neutron remains unchanged when it interacts with a nucleus, the scattering is elastic. If a change in the kinetic energy of the neutron occurs then the scattering is either quasi-elastic or inelastic.

In an (elastic, coherent) neutron experiment such as small-angle neutron scattering, neutron reflection, or within the various neutron diffraction geometries, the intensity with which neutrons are scattered from a sample is measured at a variety of angles. The resulting scattering curves or diffraction distributions provide structural information on the system in question. In inelastic scattering studies, the energy changes which result from interaction between neutrons and nuclei within the sample are also measured so that an energy spectrum of scattered neutrons is obtained at each scattering angle.

In general, two factors may determine the strength with which neutrons are scattered from nuclei. These are the isotopic scattering length b and the spin-dependent (or polarization-dependent) scattering length, B. The B component is averaged out in the absence of spin polarization and therefore does not contribute to the measured signal (there are a few significant biological experiments which have made use of spin-polarization). For ²H B = 5.7 fm while for ¹H, B = 58.24 fm; hence the scattering signals from the two isotopes of hydrogen are well differentiated. This allows the scattering from a hydrogenated component of a scattering species to be deconvoluted from the deuterated whole, permitting the measurement of scattering from components representing ~1% by mass of a particle.

With b the scattering lengths of ²H and ¹H are again very different, but in this instance they are reversed so that neutrons are scattered much more strongly from ²H than from ¹H. The main reason why neutrons can be useful for the study of biological molecules is that the scattering lengths of ²H and ¹H are also opposite in sign (6.671 fm and -3.742 fm respectively) and between them almost encompass the range observed for all atoms. This means that by modulation of the ratio of ²H:¹H in a buffer the scattering from different kinds of macromolecule can be "matched out", i.e. their contrast is matched by the background scatter and so they do not contribute to the measured signal. For example, nucleic acid is matched out at ~65% ²H₂O, protein at ~42% ²H₂O, and lipids at ~13% ²H₂O. Additionally, the match point of a molecule or components of it may be manipulated by selective deuteration.

Neutron scattering techniques can be used to investigate structures at a range of resolutions. Small-angle neutron scattering (SANS) can provide information in the range 10 to 1000 Å; specular neutron reflection and off-specular diffraction yield data in the range ~5 to 1000 Å; lamellar diffraction can yield data at comparable resolutions to X-ray crystallograpy (albeit anisotropically), as of course can neutron single crystal diffraction. The resolution range for data obtained from neutron fibre diffraction is the same as from X-ray fibre diffraction.

crystallography
 

fibre diffraction

small-angle neutron scattering and associated modelling
 

specular reflection and off-specular diffraction
 

lamellar diffraction
 

incoherent neutron scattering
 

small molecule structure determination

The ILL web page on biological neutron research and the main College VIII (biology) page
 

The European Neutron Scattering Association
 

The neutron scattering pages at Argonne National Laboratory
 

The Oak Ridge National Laboratory high flux isotope reactor

These pages have been compiled by Robert Gilbert. They are intended to describe a broad selection of the kind of biological research to which neutrons can be applied. All corrections, suggestions, offers of material etc. are very welcome. Last update 30th August 2002.

The advice and help of the following people is gratefully acknowledged: Olwyn Byron, Richard Heenan, Tony Watts, Trevor Forsyth, Dmitri Svergun, Peter Timmins .........

Division of Structural Biology homepage