-- Small-Angle X-Ray Solution Scattering (SAXS) for Protein Envelope Reconstruction --

Richard Gillilan, Quan Hao (MacCHESS) and J. Gunter Grossmann (CLRC Daresury, U.K.)

Special thanks to Jerry Houghton for machine shop help
and Michael Barclay for programming

** First measurements on the new CHESS G1 line **

-> Used 1mg/ml and 14.4mg/ml lysozyme in 50mM NaOAC buffer at pH 4.5.

Experimental setup:

• G1 wiggler line with multilayer optics
• 1.2 Angstrom wavelength with ~2% bandpass
• Quantum4 2K (ADSC) detector @ ~780mm from sample cell
• 500um x 500um beam with 0.8mm guard slits
• He flight tube with Be window on sample end, .5mil mylar on detector end.
• 1mm path (80ul) sample cell courtesy of Gunter Grossmann, Daresbury U.K.
• 25um mica windows in sample cell

Extra-large beamstop was mounted directly on .5 mil mylar of flight tube
and centered on the beam using burn paper and rail-mounted microscope.
Aligning the beam, fiddling with slits and beamstops to reduce low-angle scatter took a lot of time.


Sample cell was mounted directly on standard MacCHESS camera. Translation stage
from Supper goniometer allowed full x-z alignment of cell window.

Gunter's sample cell can be cooled using a chilled water stream, but our experiments
were done at room temperature. Protein and buffer samples
were centrifuged (next time I may use .2um filter) and loaded into the
sample chamber using a Hamilton syringe. The white teflon(?) disk has several access holes at top for syringe to enter and air to exit.

Silver Behenate standard was used to determine beam center
and calibrate sample-to-detector distance and "s" scale. Wow, 7 rings:


I took 90 20-second exposures of both buffer solution and protein solution. With the very bright beam on G1, the sample had obviously denatured after 30min.

Data Processing
I used the XTM crystal modules for OpenDX package to process the CCD images prior to reconstruction. The RadialProfile module was written with the help of RET Michael Barclay from Detroit Cody High-School.

(click to learn more)

After dark-current and geometry correction (ADSC software), I computed radial profiles by adding counts at each fixed radius for both buffer and protein solutions. Results shown here are only for one 20 sec exposure. The radial grid shown at right is part of the OpenDX user interface and shows what part of the image is being used in the calculation. The diffuse ring midway out is probably due to some kapton film that should be removed in the future.

I determined a scale factor between the sample and buffer by computing
the ratio of the two profiles and extracting the asymptotic value
of the tail (close to unity):

(r value is in pixel units)

Subtracting the scaled buffer profile from the protein solution
and plotting on the s-scale using behenate calibration, we get the scattering due to protein alone.

The momentum transfer coordinate is defined as
s = 4 pi sin(theta)/wavelength
These results are preliminary and represent only 20 sec of data!


More later. Stay tuned!