X-ray solution scattering experiments were designed according to the two-phase
m odel o f solution scattering expressed in Equation 2.5. The first requirem ent was for
a pure, monodispersed solution o f protein at a concentration that was high enough for
its scattering curve to be measured. The tendency o f protein samples to aggregate
necessitated that each sam ple was subject to gel filtration to rem ove non-specific
aggregates, then reconcentrated as shortly before data collection as possible. The choice
o f buffers is also important. In X-ray scattering experim ents, the closer the buffer electron density is to pure water, the higher the sample transm ission becomes, and hence
better counting statistics can be obtained. Phosphate buffered saline (137 m M N aC l, 2.7
mM KCl. 8.1 m M Na^HPO^, 1.5 mM KH^PO^, pH 7.5) or Tris buffer (25 mM Tris-HCl,
140 mM NaCl, 0.5 mM EOT A, 0.02% NaN^ 0.1% Pefobloc SC, pH 7.4) were used for
X-ray scattering experiments. To ensure appropriate corrections were made for solvent
scattering, the protein sample was dialysed against its buffer, and the scattering o f the buffer was subtraeted from the scattering o f the protein solution. Ideally, a sample o f
volum e 0.5 ml and concentration 2-10 mg/ml o f protein was used for X-ray experiments.
The concentration o f a protein solution can be determ ined from its tryptophan content
by m easuring its absorbance at 280 nm, and using an absorption coefficient (1%, 1cm)
calculated from its amino acid and carbohydrate com position by the corrected W etlaufer
procedure (Perkins, 1986).
2 3.2.2.
X-rav scattering at SRS DaresburvX-ray scattering experim ents were perform ed at the Synchrotron Radiation
Source (SRS) at Daresbury, W arrington, U.K. Synchrotron radiation is emitted from
stages are used in the production o f synchrotron X-rays at Daresbury (Figure 2.3a).
Electrons are produced by a hot cathode source and then accelerated to alm ost the speed
o f light in a linear accelerator (Linac). The energy o f the electrons leaving the Linac is
increased in a booster synchrotron from 12 to 600 m illion electron volts (MeV). The
electrons are then injected into the storage ring, where a high pow er radiofrequency
accelerating system increases their energy to 2,000 MeV. In the storage ring, 16 dipole
magnets force the electrons to follow a circular path, and they travel around the 96 m
circum ference 3.12 m illion times a second. The beam current at the start o f its ‘life
tim e’ is typically between 200 and 300 mA, but the current continually decreases as
electrons are lost. The electrons are usually kept in orbit for up to 24 hours before the
beam has to be regenerated. As the electrons are deflected by the magnetic field, they
em it ‘w hite’ X-rays o f all wavelengths down tangential beam lines where experimental
stations are set up to use the X-ray beams for dedicated experim ents (Figure 2.3a).
Small angle X-ray solution scattering experiments were perform ed on stations 2.1 and
8.2. A perfect Ge or Si crystal is used to horizontally focus and monochrom ate the X-
ray beam to a wavelength o f 0.154 nm. The X-ray beam is then vertically focused by a curved mirror, and collimated by sets o f slits (Figure 2.3b). This monochrom atisation
m ethod produces an X-ray beam which has negligible wavelength spread (Towns-
A ndrew et a/., 1989). Experim ents were perform ed w ith beam currents in a range o f
100-250 mA and a ring energy o f 2.0 GeV.
Samples were placed in a specially designed Perspex cell w ith 10 to 20 pm thick
ruby m ica window s (Figure 2.4a). The ruby m ica windows were held in place with
Teflon plugs, and the windows were regularly changed during data collection sessions.
The sam ple cell had a 1 mm path length, a surface area o f 2 m m (vertical) by 8 mm
(horizontal) and holds a m axim um volume o f 25 pi. The sample cell was positioned in
the X -ray beam by a brass sample holder (Figure 2.4a) w hich was m aintained at 15°C
using a w ater bath. Prior to data collection, the sample holder was aligned in the beam
using X -ray sensitive ‘green paper’ (Detex® ) that turns red on exposure to X-rays. The
scattering intensities o f the sample were measured using a 500-channel quadrant detector
Figure 2.3. X-ray solution scattering at the SRS Daresbury.
{a) Schematic representation o f the synchrotron. The linear accelerator
(LINAC), booster synchrotron and the storage ring are shown. Synchrotron radiation is emitted by electrons in the storage ring which are transm itted dow n beam lines to the different experim ental stations. The small-angle scattering instrum ents are located on beam lines 2 and 8. (A dapted from the Daresbury Laboratory W orld W ide W eb Site
http://ww w . d l ac. uk).
(b) Schem atic layout o f the X-ray solution scattering cam era at Station 2.1 at
SRS Daresbury. (Taken from http./srs.dl.ac.uk). The cam era operates at w avelength
0.154 nm using a m onochrom ator-m irror optical system. A focal spot o f size 0.3 x 2.2
mm^ is produced, w ith a beam cross-section o f 1 x
5
m m at the sample position. Theoptics are in vacuum and built on a vibration-isolation system. Sections o f vacuum tubing o f length betw een 0.5 and 5 m are m ounted on an optical bench between the sample and the detector. The scattering pattern is m easured using an area detector that is interfaced to a m inicom puter. (Taken from the Daresbury Laboratory W orld W ide
(a)
^B ooster
I M * iM tLinac
(b)
SRS Station 2.1Schem atic Floor Plan (not to scale) Monochromator diu 1 & 2 Mirror d its 3 & 4 dii8 3 & 6 S r sr r h P *l»t A Station dtUlet d its 7 6 8 f r o r t i o n cham ber Saf9ïï>i9
b ack to n cham ber
c a m era tube j Pk^^box\
Motor C ontrol ^slem ^stairs) Stairs leading to Data Anafysis Room Acquisition System D ata Collection
i
E x p ü H a tc h P r e p a r a tio n optic cl b e n c h 39Figure 2.4. a) A photograph show ing the specially designed sample holder used to m ount on the instrum ent. C represents the sample cell w hich has a 1 m m path length, a surface area o f 2 m m (vertical) by 8 m m (horizontal), and could hold a m axim um volum e o f 25 pi. The sample is injected through the injection hole and any surplus sample overflow s out o f the riser depicted by the black arrow adjacent to the injection hole. The sample is contained betw een 10 to 20 pm thick ruby m ica windows. The m ica w indow s w ere regularly changed during data collection and are held in place w ith teflon plugs. The sam ple cell was held in the X -ray beam by a brass sample holder, which w as m aintained at 15°C using coolant flowing through the tubing from a w ater bath. The tem perature is monitored by a therm ocouple inserted at T. The lever L is used to m ove the sam ple holder as depicted by the dotted white arrow to ease the insertion and rem oval o f the sam ple cell in betw een data collection. (Adapted from Perkins,
2001).
b) The scattering intensities o f the sample w ere m easured using a 500-channel quadrant detector. A quadrant detector m easures intensities in a 70° angular sector o f
a circle, and gives good counting statistics at large Q values where the intensities are
a)
Injection hole
Coolant flow tubing
Ruby mica
window Teflon plug
angular sector o f a circle, and gives good counting statistics at large Q values where the intensities are weaker. The nominal position o f the m ain beam is located at the centre
o f the circle, and a beam stop made from lead was used to protect the detector. The
response o f the 500 detector channels is not uniform, so for each data collection session
the detector response was measured for several hours using a uniform ^^Fe radioactive
source, and this was used to correct the experimental X-ray scattering measurements
(Figure 2.5a). The distance between the detector and the sample was set so that
intensities were measured to a maximum Q value o f 2.0 to 2.2 n m '\ On station 2.1 sam ple-to-detector distances o f 3.325 m to 5.635 m were used (the longer cam era length
can be seen in Figure 2.6a) while on station 8.2 a sam ple-to-detector distance o f 3.59 m
was used. Scattering intensities were measured as a function o f detector channel
num ber, so for each data collection session the X-ray diffraction pattern o f fresh, wet,
slightly stretched rat tail collagen was measured for calibrating the Q range based on a
diffraction spacing o f 67.0 nm (Fraser and MacRae, 1981; Figure 2.5b). X-rays produce free radicals that can be destructive to proteins, causing them to aggregate quickly on exposure. To check for this possibility, the scattering intensity o f each sample was
m easured for 10 minutes in 10 equal time frames, and the tim e frames were inspected
for radiation-dam age effects. The protein samples were measured in alternation with
their respective buffers in order to m inim ize buffer subtraction errors as the incident
beam decreased in intensity. For each sample, an ion cham ber m onitor positioned before
the sam ple holder was used for m onitoring o f the incident X -ray beam intensity, while
m easurem ents from a second ion cham ber m onitor positioned after the sample holder
autom atically allowed for the sample transm ission and incident flux in data reduction.
2.3.2.3. Reduction o f SRS scattering data
SRS scattering data were reduced to obtain scattering curves I(Q). The SRS
scattering data were w ritten as binary files, which contained ten tim e frames o f scattering
intensity as a function o f detector channel number. D ata reduction was perform ed using
the OTOKO software (Figure 2.6b; Bendell, P., Bordas, J., Koch, M .H.C., and Mant,
G.R., EM BL Hamburg and CLRC Daresbury Laboratory, unpublished software). All
scattering data were normalised to the counts measured by the ion cham ber positioned after the sample holder using the .DIN procedure. This corrected for beam flux,
6 x 1 0 * jO C 3 O ü C