RESOURCES

Name of Method: SAXS

Result expected: Small-angle X-ray scattering (SAXS) allows one to study native biological macromolecules, from individual proteins to large complexes, in solution under nearly physiological conditions. SAXS not only provides low resolution three-dimensional models of particle shapes but yields answers to important functional questions. Kinetic SAXS experiments allow one to analyze structural changes in response to variations in external conditions, protein-protein and protein-ligand interactions, and to study kinetics of assembly/dissociation or folding/unfolding.

K_D range:
Can characterize interactions with K_D more than ......... and less than 10^-3 µM

MW range: 5 kDa - 200kDa

Purity: The samples should be monodisperse (desirably > 90%).

Labeling-Tagging: Should any or all of reactants be labeled or tagged and how? Not necessary

Accuracy: (Da) 3-5 kDa

Quantity required: For a complex AB, assuming that A is the 'receptor' and B the 'ligand' how much protein is needed to characterize the complex fully (e.g. obtain K_D or get MW) ?
A total of 100÷¸300 µl of A, at a concentration of 2÷20 mg/ml, total of 2÷5 mg of A.
A total of 100÷¸300 µl of B at a concentration of 2÷20 mg/ml, total of 2÷5 mg of B.
A total of 100÷¸300 µl of AB at a concentration of 1÷10 mg/ml, total of 1÷3 mg of AB.
Assume a MW of about 50k_D to convert moles and M to mg and mg/ml
... for that MW, 1mg/ml is 20 µM ... only fill what is relevant for the method.

HTP: Is the method HTP (can it use 96-well sample plates in an automated fashion)? Yes

Speed: Will the user have a quantitative answer immediately after collecting the data? Yes

Cost: (approximate cost of consumables per ONE experiment) Free for academic users,
proposals are evaluated by EMBL Hamburg Priorities Committee

Access: Is an instrument available (name all SPINE2 sites you know)? EMBL, Hamburg Outstation, X33 beamline

User expertise: Can the method be provided as a service (measurement and data analysis done by local staff) or only access and assistance on a collaborative basis? Both

Special requirements: Any special sample needs?
96-well plate sample changer will be on-line in Hamburg in September 2007 - this will allow measurements in 305 minutes per sample and this can be coupled with an HPLC.
SAXS characterization of protein complexes will be the subject of a SPINE 2 workshop - dates tba.