Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 150

Two basic types of X-ray sources are available for collection of SAXS data:
synchrotron radiation and bench-top sources. While synchrotron radiation provides a
higher intensity of X-rays and thus reduces data collection time, it can also increase
the risk of radiation damage to the sample. To minimize radiation damage, the sample
can be flowed back and forth across the beam during data collection. Secondary radia-
tion damage can also occur due to X-ray-induced generation of hydroxyl radicals,
which can promote RNA hydrolysis. To minimize this, tris buffer (~20 mM) can be
included as an effective hydroxyl radical scavenger. Collection of SAXS data using a
bench-top instrument requires a smaller sample volume (as little as 30 m L); however,
the low intensity of the X-rays requires a much longer data collection time. While it is
tempting to overcome this problem by using a higher concentration of RNA, this may
result in interparticle repulsion as described above (Putnam et al. 2007 ) .

8.1.3.2 Interpreting SAXS Data

The SAXS pro fi le, I ( q ), results from observation of X-ray scattering from all orien-
tations of the molecule in solution (Rambo and Tainer 2010b ) . The resulting 2D
scattering pattern can therefore be radially averaged and converted into a 1D scat-
tering curve to maximize the amount of signal obtained from a given experiment.
The scattering curve contains valuable information about the dimensions, volume, and
fold of an RNA molecule (Fig. 8.7a ). The scattering angle is expressed in reciprocal
space as a function of q (Koch et al. 2003 ) :

4sin πθ

λ

q =

(8.1)

where q is one-half the scattering angle from the incident beam and l is the wave-
length of the X-ray radiation. The maximum intensity of the scattering curve is
dependent on the source and type of detector and is therefore frequently normalized
to 1 to allow for comparison of different data sets. The smallest angles provide infor-
mation about the size of the molecule (Fig. 8.7a ). For most molecules, this falls in the
range of q < 0.05 Å −1 . Scattering in the range of q < 0.3 Å −1 contains information
about the shape of the molecule. Peaks observed in the range of 0.3 < q < 1.0 Å −1 arise
from internal secondary structure within the molecule (Fig. 8.7a ). Unfortunately, due
to the fact that the molecules in solution are randomly oriented, high resolution infor-
mation cannot be extracted from this region for the purposes of structure analysis.
The radius of gyration ( R g ) is the root mean square distance of electron density
from the center of mass and provides an accurate measure of size and shape that is
useful for comparison between different samples. R g can be estimated using a
Guinier transform which exploits an approximately linear relationship between
ln( I ( q )) and q 2 at low values of q (Konarev et al. 2003 ) :

1

2

ln ( ) Iq =− I (0) Rq (8.2)

g

3