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D22

Large dynamic range small-angle diffractometer

Call for projects - Small Angle NEUTRON Scattering for Membrane proteins

Free trial | New capabilities dedicated to the structural study of membrane protein in solution

What are these new capabilities?

Beside crystallography and cryoEM, structural biology also include low resolution (about 1 nm) techniques in solution named Small angle scattering (SAS). They can be applied to samples that are too flexible to crystallize, to macromolecular complexes or to conformational equilibrium. Their results can be analyzed ab initio or by fitting atomic model, and provide large-scale information complementary to high-resolution techniques to understand protein behavior in solution.
The probe used in SAS techniques can be an X-ray Beam (SAXS) or a Neutron beam (SANS). SAXS is very powerful for soluble protein but cannot distinguish between molecules of different nature, such as protein and detergent, while SANS can: using specific deuteration, the detergent can be made invisible to the neutron beam. In consequence, SANS is adapted to the study of membrane protein in solution. This is known for decades, but until now, several technical constraints blocked the generalization of the technique: 1/Detergents adequately deuterated were difficult to obtain, 2/Neutron sources had insufficient flux, 3/Instrumental set up and sample environment were not adapted to biological samples.
These technical locks have been open and today, the Australian and European neutron sources (ANSTO and ILL) are happy to offer:

Neutron sources are large instruments open to users according to scientific excellence of their experimental proposal(s). But new technical capabilities require to build a new community of users. Many membrane protein specialists did not mind acquiring SANS expertise up to now, because this technique was useless for them. Therefore, to help building this community, we propose a trial session open to any scientist of the academic sector within requirements listed below.

If you are wondering which conformation your favorite membrane protein adopts in solution, you can send us one or two samples to be analyzed by SANS.

We will return experimental data and help in analyzing them if necessary. If you are interested in continuing such study, you will be able to include these results in experimental proposals, establishing the project feasibility. For this free trial, ILL and ANSTO are sharing the cost of detergent deuteration and D2O. For further studies, these fees linked to sample preparation will be on you.

Today a subset of detergents is available with the adequate deuteration scheme, so only the protein solubilized in ß-DDM, ß-OG or LMNG can be accepted. Depending on requests from your side this subset may be expanded in the future.

Technically, one SEC-SANS analysis will requires you to send the following:

And then, if the project is feasible,

  • A sample of 200µL of protein at minimum 2 mg/mL.
  • The powder resulting from freeze-drying 150 mL of buffer without detergent.
  • Your lab must be in one of the ILL member countries: France, Germany, United Kingdom, Spain, Switzerland, Austria, Italy, the Czech Republic, Sweden, Belgium, Slovakia, Denmark, Slovenia or Poland.
  • When publishing the results, people involved in deuterated detergent synthesis or SANS data recording and analysis must be included in the author list (maximum 4 names). The facilities should be acknowledged, and the DOI of the data cited.
  • You have to agree with ILL data policy stating that data should be published and the raw SANS data will anyway be released to the public 2 years after the measurement.
  • Before sending any sample, please send your project description (pdf - 271 Ki) to Anne Martel to ensure feasibility.
  • She will perform the experiment by herself but your remote participation would help ensuring its success.

If a selection of projects is required, the novice neutron users will be favored.

  • A data bank of biological SAS data exists (SASBDB) and we advice to deposit your results there to help further development of analysis tools.
  • IUCr edited guidelines (pdf - 1.33 Mi) for Bio-SAS data publication.
  • The detergent-free buffer powder will be dissolved in 150 mL of D2O, deuterated detergent will be added and the solution will be filtered and degassed. It will be used to equilibrate the column and elute the protein.
  • The protein will be injected on a Superdex column (SD75 or SD200 depending on its size) and eluted with the buffer at sufficiently low flowrate to enable the exchange of the detergent initially present in your sample, including the fraction bound to the protein, for deuterated one present in elution buffer.
  • The chromatogram will be recorded at 4 wavelengths. One neutron scattering pattern will be recorded every 30 s through the whole elution, using optimized SANS instrument set up. Scattering patterns will be reduced. You will be involved in their analysis by video-conference.