NEUTRON CRYSTALLOGRAPHY OF BIOLOGICAL MOLECULES
E.
Buchtelová
Department of
Biochemistry, Faculty of Science, Charles University, 128 43 Prague, Czech
Republic
Advantages
of neutron diffraction - why to use neutrons
Neutron diffraction is used much less than X-ray diffraction for protein
structure determination. This is because the flux of neutron beams is much
lower than the flux of X-ray beams, and also because of low availability of
neutron sources. Nevertheless some problems can be solved only by diffraction
of neutrons [1]. The main advantage of neutrons is their relatively high
elastic scattering by hydrogen. Hydrogen atoms can be seen in maps obtained
with neutrons even if they are not visible with X-rays. Neutron diffraction is
therefore used to determine the positions of important hydrogen atoms, such as
atoms involved in enzymatic reactions or in important hydrogen bonds and to
study hydratation of biomolecules. Neutron diffraction can tell us whether a
particular acidic group is dissociated or has a hydrogen atom bound to it, it
can discriminate between water and hydroxyl anion in the active site of an
enzyme, it can determine the orientation of a water molecule. Even if only low
resolution data can be obtained, neutron diffraction can give us information on
the position of solvent or other hydrogen containing molecule. In such cases
labelling of the molecule of interest by replacing its hydrogen atoms by
deuterium is used.
In contrast to X-rays, neutrons are not scattered by electrons but by
the nuclei of atoms in biological materials. The interactions of neutrons with
nuclei depend on strong nuclear forces and the scattering amplitudes vary from
element to element in a non systematic way, which means atoms of similar atomic
mass can be distinguished which would seem the same with X-rays. Even different
isotopes or nuclei with different spin have different scattering amplitudes.
The scattering amplitude of
hydrogen is of the same order of magnitude as the amplitudes of other atoms
predominating in biological molecules (Table 1.), so the scattering by hydrogen
is not overridden by heavier atoms. The scattering from hydrogen can be
increased even more by replacing hydrogen H11 by
deuterium (H12, often written as D). The deuterium
amplitude is almost twice as high as that of hydrogen H11.
Moreover incoherent scattering, which creates background in the diffraction
image, is lower for deuterium than for hydrogen. The deuterium amplitude is
positive, while the hydrogen H11 amplitude is negative.
When hydrogen atom is bound to a heavier atom, the neighbouring positive and
negative densities can partially cancel out. This does not happen with
deuterium. The difference between hydrogen and deuterium scattering can be used
for labelling only some atoms and/or computing difference in maps from data
obtained with hydrogen only and from data with deuterated molecule(s). By
comparison of the maps the position of the deuterated molecule can be assigned
to a peak in positive difference map even if the resolution is too low to
recognise it from the shape of the peak.
Table 1. Coherent neutron scattering amplitudes [2].
|
Element
or isotope |
H |
D |
C |
N |
O |
|
Coherent
neutron scattering length bcoh (10-15m) |
-3.7 |
6.7 |
6.6 |
9.4 |
5.8 |
The easiest and cheapest way to partially deuterate the crystal is to
incubate it in D2O. Protein crystals have a large content of solvent
H2O, which will be replaced by D2O, and some of the
hydrogen atoms of the protein or other biomolecule will exchange with the
deuterium of the solvent. If we want to obtain a completely deuterated
biomolecule, we have to grow the cells in which the biomolecules are
synthesised in D2O and supply them with deuterated food. That is much more expensive.
By using different ratios of H2O to D2O for the
solvent it is possible to enhance contribution of specific parts of the crystal
(for example detergent, lipids, nucleic acids or deuterated protein) to the
scattering at low resolution and so to determine at very low resolution even
the structure of disordered parts of the crystal.
Disadvantages
of neutron diffraction - experimental limitations
The main disadvantage of neutrons is the low flux of the beam. At the
LADI (Laue diffractometer) experimental station at ILL, the largest existing
neutron beam source, the flux of partially monochromatised beam (l = 3.5 Å; dl/l = 20%) is 3 x 107 neutrons.cm-2.s-1,
which is about ten orders of magnitude lower than the flux of monochromatised
beams from whigler sources on synchrotrons. The strategy of using a partially
monochromatised beam and quasi-Laue difraction pattern for structure
determination was adopted at the LADI experimental station in order to decrease
data collection time. Even so the data collection
takes at least two weeks compared to less than hour at the synchrotrons with
the highest flux.
Because of the low flux and low interaction of neutrons with the sample
large samples are necessary. The smallest protein crystals usable at present
for neutron diffraction have to have volume 1 mm3. The crystals
diffract to about 1 Å lower resolution than they do with X-rays. If the purpose
of the experiment is to see the hydrogen atoms, the lowest resolution which
gives acceptable results is around 2.8 Å, so only reasonably well ordered
crystals can be used. Large size of molecule also hinders the experiments - the
molecular weight of the largest protein for which data have been collected at
LADI was 50 000 Da.
Description
of the LADI experimental station at ILL
The LADI experimental station is mainly used for single-crystal studies
of proteins at medium or high resolution [3]. It uses cold neutrons (neutrons
with long wavelength). The long wavelengths, around 3.5 Å, have been selected
to increase the scattered intensities. As mentioned before, a broad bandwidth
of wavelengths is used combined with quasi-Laue structure determination. A
still image is taken and than the crystal is rotated to the next position (for
example by 8°) to cover the whole reciprocal space. A detector based on the
image plate technology has been designed for this experimental station. It has
been adapted for detection of neutrons by incorporating of Gd2O3
into the image plate. When hit by a neutron, Gd emits X-rays, which are then
detected in the image plate in the normal way, first being absorbed by the
colour centres to form a latent image, which is then read out by exposure to
visible laser radiation. The detector is cylindrical, the sample being placed
in the centre of the detector. Thus reflections diffracted to high angles can
be recorded, which is necessary with the long wavelengths that are used. The
resolution limit of the detector using the above mentioned wavelengths is
around 1.5 Å. For the crystals that have been measured so far this limit was
sufficient - none of them, with the exception of test lysozyme, diffracted to
such a high resolution. For crystals with larger unit cell spatial overlaps can
be a problem. This can be solved by decreasing the range of wavelengths to
which the crystal is exposed and by decreasing the angle of rotation of the
crystal at the same time. For extremely large unit cells (viruses) a planar
detector perpendicular to the beam is also available.
The sample is usually placed in a capillary and measured at room
temperature. Neutrons do not cause radiation damage of the protein crystal. If
lower temperatures are of advantage, for example for decreasing the temperature
factors of the solvent, cooling by liquid helium can also be used.
For data treatment software based on CCP4 Laue suit is used [4]. The
indexing of the diffraction pattern takes advantage of the fact that cell
parameters are already approximately known from diffraction experiments with
X-rays. The main problem in data treatment is normalisation of the data for
spectral distribution of the neutron beam, that is, reflections have to be
scaled according to the intensity of the part of the source beam with
wavelength that fulfilled diffraction conditions.
Once appropriate intensities have been assigned to the reflections,
structure determination proceeds as is usual in X-ray protein crystallography,
with the exception that neutron scattering amplitudes are used instead of X-ray
scattering factors.
Examples
of usage of neutrons in biocrystallography
The structure of aspartic protease endothiapepsin complexed with an
inhibitor was determined by Laue diffraction study at LADI experimental station
at 2.1 Å resolution [5]. The protonation state of catalytic aspartates was
determined: Asp 215 was found to be protonated and Asp 32 was not protonated.
The neutron structure of hen egg white lysozyme at pH 7.0 determined to
2.0 Å resolution at LADI experimental station showed that neither of the
catalytic residues Glu 35 and Asp 52 was protonated at pH 7.0, while earlier
neutron studies had shown that Glu 35 is protonated at pH 5.0 [6].
The position of deuterium atoms of the bound water in concanavalin A was
determined at 2.4 Å resolution by Laue diffraction. The authors were mainly
interested in water bound in the saccharide binding site and in the metal
binding site [7].
The hydrogen bonding network of cellulose II was determined by neutron
fibre diffraction [8].
The hydratation of DNA in A
conformation was determined by neutron fiber diffraction [9]. Two chains of
water were found in major groove, one chain linked the oxygen atoms of the
phosphate backbone and a water feature was found in the centre of the molecule
which was not resolved in the direction of the axis of DNA and which is
supposed to be sequence-dependent.
[1]
E. Pebay-Peyroula & D. Myles: Neutron crystallography of biological
molecules. In: Structure and Dynamics of Biomolecules: Neutron and Synchrotron
Radiation for Condensed Matter Studies, HERCULES volume IV, Oxford University
Press, 2000.
[2]
R. Scherm, Ann. Phys., 7 (1972)
349-370.
[3]
http://www.ill.fr/YellowBook/LADI/
[4]
http://www.ill.fr/YellowBook/LADI/ladimanual/Lmain.html
[5]
L. Coates, P.T. Erskine, S.P. Wood, D.A. Myles & J.B. Cooper, Biochemistry, 40 (2001) 13149-13157.
[6]
N. Niimura, Y. Minezaki, T. Nonaka, J.C. Castagna, F. Cipriani, P. Hoghoj, M.S.
Lehmann & C. Wilkinson, Nature
Struct. Biol., 4, 909-917.
[7]
J. Habash, J. Raftery, R. Nuttall, H. J. Price, C. Wilkinson, A.J. Kalb
(Gilboa) & J.R. Helliwell, Acta
Cryst. D, 56 (2000) 541-550.
[8]
P. Langan, Y. Nishiyama & H. Chanzy, J.
Am. Chem. Soc., 121 (1999) 9940-9946.
[9]
M.W. Shotton, L.H. Pope, T. Forsyth, P. Langan, R.C. Denny, U. Giesen, M.T.
Dauvergne, & W. Fuller, Biophys Chem., 69 (1997) 85-96.