research papers
A high-performance neutron diffractometer for
biological crystallography (BIX-3)
Journal of
Applied
Crystallography
ISSN 0021-8898
I. Tanaka, K. Kurihara, T. Chatake and N. Niimura*
Received 27 June 2001
Accepted 18 October 2001
Advanced Science Research Center, JAERI, Tokai, Ibaraki 319-1195, Japan. Correspondence e-mail:
[email protected]
# 2002 International Union of Crystallography
Printed in Great Britain ± all rights reserved
A high-performance neutron diffractometer for biological crystallography
(BIX-3) has been constructed at JRR-3M in the Japan Atomic Energy Research
Institute (JAERI) in order to determine the hydrogen-atom positions in
biological macromolecules. It uses several recent technical innovations, such as a
neutron imaging plate and an elastically bent silicon monochromator developed
by the authors. These have made it possible to realise a compact vertical
arrangement of the diffractometer. Diffraction data have been collected from
the proteins rubredoxin and myoglobin in about one month, to a resolution of
Ê . The data were good enough to identify the hydrogen atoms with high
1.5 A
accuracy. By adopting a crystal-step scan method for measuring Bragg
diffraction intensities, the signal-to-noise ratio was much better than that of
the Laue method. This shows that BIX-3 is one of the best-performing machines
for neutron protein crystallography in the world today.
1. Introduction
The three-dimensional structures of proteins and other biological macromolecules have been determined mainly by X-ray
crystal structure analysis, which has made extraordinary
contributions to the life sciences. Such results have clearly
suggested that hydrogen atoms and the water molecules
located around proteins and DNA may play a very important
role in many physiological functions (Westhof, 1993; Desiraju
& Steiner, 1999; Jeffrey & Saenger, 1991). However, since it is
very dif®cult to determine the positions of hydrogen atoms in
protein molecules using X-rays alone, a detailed discussion of
protonation and hydration sites can currently only be speculative. In contrast, it is very well known that neutron
diffraction provides an experimental method for the direct
location of hydrogen atoms (Niimura, 1999). Unfortunately, to
date there are relatively few examples of the use of neutron
crystallography in biology, because a considerable amount of
time is needed to collect a suf®cient number of Bragg re¯ections. In order to overcome such dif®culties, a neutron
diffractometer for biological crystallography (BIX-3) has been
designed (Niimura, 1999; Tanaka, Kurihara et al., 1999) and
constructed (Tanaka et al., 2001) at the JRR-3M reactor in the
Japan Atomic Energy Research Institute (JAERI). Several
interesting results (Niimura, 2001) will be published soon as
full papers.
At the largest research reactor in the world, the Institute of
Laue-Langevin (ILL) in France, a quasi-Laue neutron
diffractometer for biological crystallography (LADI) has been
constructed (Wilkinson & Lehmann, 1991; Cipriani et al., 1995;
Myles et al., 1998) and some results have recently been
reported (Niimura et al., 1997; Langan et al., 1999; Bon et al.,
1999; Cooper & Myles, 2000; Habash et al., 2000). The merit of
34
I. Tanaka et al.
High-performance neutron diffractometer
the Laue diffraction technique is the high speed in collecting
data, based partly on the fact that a broad range of wavelengths is used. But the Laue method has some inherent
disadvantages, namely a poor signal-to-noise ratio arising from
incoherent background radiation, and severe overlap of
re¯ections due to the fact that a large area is being scanned at
once. Such problems are much less severe with an instrument
equipped with a monochromator, such as BIX-3 at JAERI.
Prior to the construction of BIX-3, two other machines had
been operational at JRR-3M in JAERI. One was the fourcircle diffractometer BIX-1, installed at the same port (1G-A)
as BIX-3 in the reactor hall (Niimura, Tanaka, Minezaki et al.,
1995). It was constructed with the purpose of starting a project
in Japan to study biological macromolecules with neutron
crystallography. This project also involved the development of
a new type of an elastically bent perfect silicon (EBP-Si)
monochromator (Niimura, Tanaka, Karasawa et al., 1995).
However, there was an inevitable problem with the detector.
Even though conventional 3He detectors at BIX-1 were
adequate for the analysis of re¯ections from crystals with small
unit cells (Ohhara et al., 1998; Tanaka, Ohhara et al., 1999;
Ohashi et al., 1999; Ohhara et al., 2000, 2001), for protein
crystals the identi®cation of re¯ections between nearestneighbour Bragg spots was ambiguous, because of the insuf®cient linearity in spatial resolution (Tanaka et al., 1998), and
this made proper indexing impossible. To overcome the
problem, a neutron imaging plate (NIP) detector was designed
by the authors (Niimura et al., 1994). This new -sensitive
neutron detector was originally applied to another diffractometer for biological crystallography (BIX-2), constructed at
the thermal guide hall in JRR-3M (T2-3). The location of
BIX-2, situated much further away from the reactor core than
J. Appl. Cryst. (2002). 35, 34±40
research papers
BIX-1, was selected because of the lower -ray background
level at that site (Fujiwara et al., 1998). However, with BIX-2 it
took more than 100 days to collect a complete data set from a
protein crystal (Maeda et al., 2001) because of the low neutron
¯ux at that remote location, despite much efforts at improving
intensity via a redesigned monochromator. Finally, the authors
decided to replace BIX-1 and construct a new diffractometer
with an NIP detector (BIX-3) in the reactor hall.
However, placing the instrument back at the original position closer to the reactor core meant that, while the neutron
¯ux is higher, the unwanted -ray background radiation was
also higher. Fortunately, it turned out that after careful
adjustment of the -ray shielding and the neutron shielding at
the planned site, the NIP detector could be ef®ciently
protected, even in the reactor hall, by considering the energy
dependence of the -ray sensitivity of the NIP (Tanaka,
Kurihara et al., 1999; Tanaka et al., 2001; Haga et al., 1999). As
a result, BIX-3 has three novel distinguishing features: (a) an
unconventional monochromator design (EBP-Si), (b) an
evolutionally excellent NIP detector, and (c) a vertical
arrangement of instrument components. The ®rst feature, the
EBP-Si monochromator, has made it possible to focus a beam
of monochromated neutrons on a relatively small sample area.
The second feature, the neutron imaging plate, has allowed a
more ef®cient recording of re¯ections and has enabled a larger
solid angle to be subtended. The third feature, the vertical
design of the instrument, has helped to minimize the ¯oor area
occupied by BIX-3 and to allow the sample position to be
closer to the monochromator. BIX-3 can complete data
collection in about one month on a crystal several cubic
Ê.
millimetres in volume with a resolution of 1.5 A
In this paper, the construction of BIX-3 is described, the
high-performance capabilities of instruments are discussed,
and a comparison is made between BIX-3 and the LADI
instrument at ILL.
2. Technical aims and necessary requirements
Among the different applications of neutron diffraction,
protein crystallography is one of the most dif®cult experimentally because of the weakness of the diffraction pattern
from biological samples. In order to overcome this dif®culty,
the three following items have been proposed and developed
(Niimura, Tanaka, Minezaki et al., 1995). (i) To increase the
intensity at the sample position by focusing the neutron beam
to a smaller cross section (5 mm2), which is acceptable because
the typical size of protein single crystals is at the most several
mm3; (ii) to establish a systematic approach to grow protein
single crystals up to the size of several mm3; (iii) to develop a
high-performance neutron detector to cover a large area with
a higher signal-to-noise ratio, higher spatial accuracy and
better neutron detection ef®ciency. Among these, the second
item is beyond the scope of this paper and will not be
discussed here. The other items will be discussed in detail in
the following section of this paper (x3).
The construction of a neutron diffractometer for biological
macromolecules would be practical only if its performance
could satisfy the following two criteria. (a) It has to be able to
separate nearest-neighbour Bragg spots on the detector while
step-scanning, and it has to produce frames which can be
indexed and integrated properly, when generated by crystals
Ê in the maximum dimension.
having unit cells of up to 90 A
Ê -resolution data
(b) It has to complete the collection of a 2 A
set in about one month.
3. Design
Figure 1
A photograph of BIX-3 without the detector shield.
J. Appl. Cryst. (2002). 35, 34±40
The most characteristic and novel design is the vertical
arrangement of the main components of the diffractometer
(Fig. 1). The vertical design simultaneously solved the low-¯ux
problem by allowing the sample position to be closer to the
monochromator, as well as the shielding problems by enabling
a suf®ciently small quasi-cylindrical detector shield to be built.
Most importantly, the vertical arrangement dramatically
decreased the overall size of the diffractometer, thereby
eliminating space-sharing problems with neighbouring
machines.
The positioning and orientation of the sample is also a
notable design feature (Fig. 2). The goniometer with a sample
crystal is set in an unconventional upside-down position, in
order to permit access and allow rod rotation by the NIP
reader. In order to maintain simplicity around the actual
sample mount, crystal alignment is carried out with an alignI. Tanaka et al.
High-performance neutron diffractometer
35
research papers
3.1. Monochromator
In order to obtain more intensity for the biomacromolecular sample, the size of which is 10 mm3 at the most, an
elastically bent perfect silicon crystal (EBP-Si), adjusted by a
piano-wire tension device, has been developed and found to
be very effective (Niimura, Tanaka, Karasawa et al., 1995;
Tanaka, Niimura & Mikula, 1999). It has the ability to focus
the neutron beam onto the sample position. Because the
bending of two silicon plates in piles has also been developed,
BIX-3 has succeeded in obtaining more neutrons at the sample
position by a factor of 1.6 compared with an optimally bent
single plate (Tanaka et al., 2000). The unit dimensions of the
silicon plate are 250 mm in width, 40 mm in height and 5 mm
in thickness.
Ê is used
As shown in Fig. 3, the longer wavelength of 2.35 A
(from a beam hole set at an angle of 2M = 44 ) in order to
satisfy the ®rst necessary requirement that the neutron beam
should be useful for larger unit cells.
3.2. Beam path from monochromator to sample and sample
condition
Figure 2
The crystal alignment tool of BIX-3.
ment tool, prior to data collection, at a remote location that
mimics the real goniometer table.
In Fig. 3, the overall layout and a schematic view are shown.
From the upstream to the downstream portion of the neutron
beam, each part of the instrument will be explained sequentially in detail.
Figure 3
The layout of BIX-3 (left) and a schematic view of the instrument (right).
36
I. Tanaka et al.
High-performance neutron diffractometer
The ¯ight tube and slit system were designed especially for
protection against -rays because of the sensitivity of the NIP
detector. They are made of lead (Pb) and natural lithium
¯uoride (LiF) tiles, respectively. Their purpose is to prevent
any leakage of -rays in the downstream direction. The sample
is set upside-down (Figs. 2 and 3). At present the measurement
environment is at room temperature and under normal
atmospheric pressure.
3.3. Detector
3.3.1. Neutron imaging plate (NIP). During the development of the NIP, while improvements were made to make it
useful in practice (Niimura et al., 1994), the problems with the
detector were gradually overcome (Niimura et al., 1997). Since
it is designed as a large ¯exible sheet,
the solid angle subtended by it around a
sample can be very wide. The highest
spatial resolution (less than 0.4 0.4 mm) enabled us to reduce the
distance between the sample and the
detector to 200 mm, as well as the
overall size of the diffractometer. But
the NIP is sensitive to the -rays, especially in the reactor hall, and thus
shielding against -rays was necessary.
This procedure will be discussed in a
later section (x3.4).
3.3.2. Reading system. The reading
system at BIX-3 is composed of a
semiconductor laser, having a wavelength of 635 nm and power (at the exit
of the laser) of 15 mW, reduced to
approximately 10 mW on the NIP
surface because of attenuation by the
lens. At the end of a rotating rod (Fig. 4)
J. Appl. Cryst. (2002). 35, 34±40
research papers
there is a window both for the laser light irradiation and also
for the detection of the photostimulated luminescence (PSL).
The reading proceeds as the NIP moves downwards, parallel
to the cylinder axis. It takes only 3 min for reading, 1 min for
erasing and 1 min for the translational movement of the NIP
itself. This total, about 5 min, is the necessary processing time
per frame between exposures. In Fig. 4, the NIP is shown as a
white cylinder.
In order to measure spot intensities accurately, the effect of
any intensity decrease as a function of elapsed time (fading)
must be evaluated and corrected for. This fading process is an
inherent feature of the PSL detecting principle and depends
upon the laser power, reading time, temperature (Amemiya &
Miyahara, 1988) and constituent materials of the NIP. From a
calculation using parameters of elapsed-time and read-intensity regression curves in fading experiments at BIX-3, using
neutron beams of 100% and 10% intensity, it was found that
the maximum intensity difference is less than 2%, whether it
was read at the beginning or at the end of the 3 min reading
time (Tanaka, 2000). In the calculation, it was assumed that
the fading starts as soon as the NIP is being exposed. The
fading strongly depends upon the laser power of a reading
system. The read intensity decreases more rapidly when the
laser power is weak (Tanaka, 2000). This effect may be
magni®ed for large imaging plates. At BIX-3, data are
presently processed without corrections of intensities, because
errors in intensity caused by other reasons are expected to be
larger than the fading error of 2%.
It can be concluded that the fading phenomenon does not
signi®cantly affect the recorded intensities, except for extremely high accuracy crystallography. Additionally, whenever
the components of the reading machine are changed, such as
the kind of NIP, the reading laser, or one of the components
involved in amplifying signals, we recommend that the
recorded intensities should be calibrated again because the
fading effect strongly depends upon the reading system.
3.4. Shielding
If the shield is in the form of a small house (i.e. a hutch-like
experimental area), the total volume of the diffractometer will
be larger and a signi®cantly lower level of incident neutrons
will be obtained because the sample position will be farther
from the monochromator. Therefore, it was decided to
construct a compact machine by designing the frame of the
diffractometer vertically and by arranging the necessary parts
carefully (Fig. 3). The shield is composed of two layers, an
exterior neutron shielding of 50 mm thickness of B4C mixed
with resin, and an interior -ray shielding, of 50 mm thickness,
made of lead. The individual background levels of neutrons
and -rays were measured and were found to be comparable.
Because of the decision to downsize the instrument, the whole
machine could approach the monochromator much more
closely and be accommodated within the biological shield (Fig.
3). As a result, the background could be shielded naturally.
Moreover, in order to shield the NIP from sample-originated -rays, it was found, by experiments at the proposed site
J. Appl. Cryst. (2002). 35, 34±40
for BIX-3 (Tanaka, Kurihara et al., 1999; Tanaka et al., 2001),
that a relatively thin piece of lead was enough to shield the
NIP (Haga et al., 1999). A lead plate of 1 mm thickness was
attached to the Al holder and was set at a distance of 30 mm
inside the cylindrical NIP, surrounding the sample (upper part
in Fig. 4). This thickness produced the best signal-to-noise
ratio of Bragg spots over background at the NIP (Haga et al.,
1999).
As the beam path of neutrons through the ¯ight tube is
under a normal atmospheric pressure, scattering from air was
not negligible. But most of it could be suppressed by coating
the inner surface of the direct-beam transporting tube with
B4C + resin, both at the upstream and the downstream positions, a procedure which contributes to a lowering of the
background at the detector.
4. Measurement method
Because measurement of diffraction patterns by oscillation
may make the signal-to-noise ratio worse, as in the Laue
method, we decided to use a step-scanning procedure as the
basic measurement method. After the data on the NIP are
read for each exposure, the sample is rotated around the
goniometer by a small angle (typically 0.3 ), and then the
erased NIP is returned to the measurement position again.
This procedure is repeated several hundred times, with each
exposure lasting from approximately half an hour to about
one hour for typical samples. It is, however, easy to make
mistake-free measurements without any investment of labour
because of the fully automated feature of the system. The
actual scanning angles depend upon the symmetry of the
crystal group of the sample. Since in most cases it is not
suf®cient to scan a sample around a single axis, it is usually
necessary to dismount the crystal, turn it by approximately 90
to obtain an alternative orientation, and then reseal it in the
same capillary tube to collect a second set of frames. In that
way, it becomes possible to collect independent re¯ections
that may be missed in the ®rst crystal orientation.
Figure 4
The image-plate reading assembly of BIX-3.
I. Tanaka et al.
High-performance neutron diffractometer
37
research papers
Table 1
Comparison of speci®cations of BIX-3 and LADI.
Diffraction principle
Incident neutron
Intensity (n sÿ1 cmÿ2)
Camera radius (sample to detector distance)
Detector, circumference longitude
Maximum re¯ection angle
Data resolution
Detector reading side
Signal-to-noise ratio
Data collection period
BIX-3 (JAERI)
LADI (ILL)
Monochromatic
Ê)
Elastically bent silicon, Si(111/311) (2.35/1.23 A
3 106 (/ = 0.015)
200 mm
Neutron imaging plate, 500 450 mm (2 plates)
143
Ê /0.6 A
Ê
1.2 A
Incident: front; data read: front
10
Ê in the case of myoglobin
22 days, dmin = 1.5 A
Quasi-Laue
Ê)
Multilayer (Ti and Ni) (3±4 A
Ê)
3 107 (/ = 0.28 at 3.5 A
159 mm
Neutron imaging plate, 200 400 mm (4 plates)
144
Ê
1.4 A
Incident: rear; data read: front
1 (as a unit)
Ê in the case of hew-lysozyme
10 days, dmin = 2.0 A
5. Data quality and discussion
The diffractometer LADI (Wilkinson & Lehmann, 1991;
Cipriani et al., 1995; Myles et al., 1998) at ILL, France, adopts a
quasi-Laue method. At ILL, one of us carried out the ®rst
neutron diffraction experiment on hen egg-white (HEW)
lysozyme (Niimura et al., 1997) using an NIP (Niimura et al.,
1994), taken to LADI from JAERI. As mentioned above, in
our opinion one can collect better diffraction data by the
monochromatic method, if possible. However, when we were
originally in the process of designing a neutron diffractometer
for biology at JRR-3M (Niimura, 1999; Tanaka, Kurihara et al.,
1999), we had to decide which method is better: the use of a
monochromator source or the quasi-Laue technique.
5.1. Principal advantage
In the true Laue method, a white beam of neutrons is used,
but in the quasi-Laue method (as used at LADI), a relatively
Ê , is used in
narrow band width, approximately from 3 to 4 A
order to try to reduce the background level and to resolve
adjacent re¯ection spots (Table 1). Its main advantages are
that the neutron intensity at the sample position is relatively
large and that many re¯ections will satisfy their Bragg
conditions at the various wavelengths contained within the
broad band. Thus, one can collect many re¯ections with one
exposure. However, this property gives rise to an inherent
disadvantage because overlap between nearest-neighbour
re¯ections easily occurs, even though complete overlap of the
higher order harmonics is inevitable, and an error may be
introduced into the calculation of integrated intensities. In
addition, the signal-to-noise ratio will be signi®cantly worse
because of background contributions from neutrons of other
wavelengths that do not satisfy Bragg conditions (Fig. 5a). On
the other hand, when monochromatic neutrons and the stepscanning method are used, Bragg re¯ections do not include
unnecessary background (Fig. 5b). As a result, the signal-tonoise ratio from a monochromatic source is much better than
that of a Laue instrument. According to a simple calculation,
the signal-to-noise ratio at BIX-3 is about ten times better
than that of LADI because the wavelength width which
contributes to a Bragg re¯ection is about 10% of the whole
wavelength band in the case of LADI. Here `noise' of `signalto-noise' means the background level. The monochromatic
incident neutron generation method de®nes such a narrow Ê ) so as to lead to a better signal-to-noise ratio (Table
(0.1 A
1). However, in the case of the monochromatic method, a huge
amount of hard-disk storage is required in order to store data
in a computer for a typical set of frames.
To summarize these comparisons, the collected data accuracy is low but the data collection speed is high in the Laue
method, and the reverse is true for the monochromatic
method. It is the aim in neutron crystallography to solve the
hydrogen-atom and bound water positions with high accuracy.
Thus, the monochromatic neutron method was adopted
because it was judged that obtaining high-accuracy data was
more important to BIX-3 than considerations of data collection time (Niimura, 1999). According to our recent experience
at BIX-3, it is possible to collect a data set that is equivalent to
the Laue case even in a short measurement time.
5.2. Advantages in the detection and recording of neutrons
on the NIP
Figure 5
Signal to noise (background) comparison. Schematic data pro®le taken
(a) by the Laue method and (b) with a monochromatic neutron beam.
38
I. Tanaka et al.
High-performance neutron diffractometer
LADI receives neutrons forming Bragg spots from a sample
crystal on the rear surface of the NIP, on which there is a
support ®lm made from a polymer (a strong scatterer of
thermal neutrons), and reads the PSL signal from the front
surface of the NIP (Table 1). This procedure loses some PSL
ef®ciency because the quantity of colour centre created by
neutrons and the penetrating depth of the reading laser on the
front surface of NIP both become less effective exponentially
as the distance from each surface increases. On the contrary, in
J. Appl. Cryst. (2002). 35, 34±40
research papers
Table 2
Details of sample crystals and experimental parameters.
BIX-3
LADI
Protein
Unit-cell dimensions
Space group
Crystal size (mm3)
Exposure time per frame
Number of frames
Myoglobin
Hew-lysozyme
Ê , = 105.8
a = 64.5, b = 30.9, c = 34.8 A
Ê
a = 64.5, c = 34.8 A
P21
P43212
6
6
25 min
12 to 24 h
999
15
is rather low and more uniform in
the monochromatic case, and that
separating re¯ections is easier. Low
backgrounds are always welcome
because weak and higher angle
intensity spots are detectable
whenever the background noise is
kept low, which leads to more reliable I(hkl) values. Uniformity of
background level may also be
expected to improve the accuracy
of the intensity measurements. Spot
separation is another important
factor. Well separated spots are
easy to integrate without errors
caused by spot overlap. All these
factors contribute to make reliable
Fourier maps in the subsequent
structural analysis.
Figure 6
A diffraction pattern from a rubredoxin crystal. Exposure time: about 1 h.
6. Summary
Figure 7
A diffraction pattern from a myoglobin crystal. Exposure time: 30 min.
the case of BIX-3, the side of the image plate which receives
neutrons is the same as the side from which the PSL is read
(Table 1), a fact which optimizes the reading ef®ciency.
5.3. Advantages in data quality
Figs. 6 and 7 are frame examples of neutron diffraction
patterns of rubredoxin (Kurihara et al., 2001) and myoglobin
(Ostermann et al., 2002), respectively, taken at BIX-3 by the
step-scan procedure. Table 2 presents details of the myoglobin
Ê in both cases.
experiment. The wavelength used was 2.35 A
The black areas at the centres of Figs. 6 and 7 are the directbeam positions. The best resolution of re¯ections is about
Ê in Fig. 6. In Fig. 7 there are re¯ections with even better
1.5 A
resolution than those in Fig. 6. Compared with the Laue
method (Niimura et al., 1997), it is found that the background
J. Appl. Cryst. (2002). 35, 34±40
From these facts, it is apparent that
the total performance of the BIX-3
diffractometer is comparable or a
little better than that of the neutron
diffractometer LADI at ILL in
France, despite the fact that ILL is
known as the highest-intensity
research reactor in the world, while
BIX-3 is situated at the mediumintensity research reactor JRR-3M.
The high performance is achieved
because key technical points were
effectively picked up, solved and
developed sequentially, and because these improvements were
integrated into the ®nal design very smoothly. At present
there are very few machines that are able to carry out neutron
data collection on protein crystals ef®ciently. However, it is
expected that the application of BIX-3 to biomacromolecules
will show that the direct determination of hydrogen atoms and
water molecules of hydration is important, and that similar
types of machines as BIX-3 will hopefully be constructed in
the future and make signi®cant contributions to important
problems in the life sciences.
The authors thank the contributions of samples used in this
study by Professor R. Bau (rubredoxin), and by Dr A.
Ostermann and Professor F. G. Parak (myoglobin). This study
I. Tanaka et al.
High-performance neutron diffractometer
39
research papers
was performed through Special Coordination Funds of the
Science and Technology Agency of the Japanese Government.
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