Method Developments
Experience from pilot structural genomics initiatives around the world shows that in the most successful case around 20% of the proteins are elucidated as structures via x-ray crystallography in the first two years. The main losses of 'efficiency' are in expression/purification and where microcrystals cannot be grown to a size suitable for data collection. The NWSGC members have a high level of collective experience in synchrotron based crystallography. Daresbury Laboratory has made pioneering contributions to SR protein crystallography in the last 20 years, see http://www.srs.ac.uk/px/publications/pub05.html. The NWSGC high intensity multipole wiggler MAD station will ensure such data collections are routine. Our main method for structure determination would be the SeMet MAD approach.
See:
A. Hdener, P. K. Matzinger, A. R. Battersby, S. McSweeney, A. W. Thompson,
A. P., Hammersley, S. J. Harrop, A. Cassetta, A. Deacon, W.N. Hunter, Y. P.,
nieh, J. Raftery, N. Hunter and J. R. Helliwell (1999) Determination of the
structure of seeno-methionine-labelled hydroxymethylbilane synthase in its active
form by multi-wavelength anomalous dispersion. Acta Crystallographica, D55,
pp. 631-643. This method is very powerful and has been applied to proteins with
up to 70 Se atoms in the structure, thus encompassing multi-macromolecular complexes.
Also:
A. Cassetta, A. M. Deacon, S. E. Ealick, J.
R. Helliwell and A. W. Thompson (1999) Development of instrumentation and methods
for MAD and structural genomics at the SRS, ESRF, CHESS and Elettra facilities. J.
Synchrotron Radiation, 6, pp. 822-833. A survey
of current known protein structures in the PDB reveals that 10% have no methionine.
Therefore, gene modification will needed. Alternative
methods will be explored in cases where this approach will not be feasible.
Phasing vehicles other than SeMet MAD can be developed such as those based on
crystallisation from sodium bromide or ammonium selenate. Phase determination
from a protein envelope (Hao, et al., 1999; Ockwell, et al., 2000)
determined using small angle x-ray scattering, along with molecular replacement
and database searches for similar protein families and structures, offer other
approaches.
Alternatively xenon and sulphur based phasing including softer X-rays (2Å) to optimise the Xe L1 and enhance the S K edge anomalous scattering respectively is now possible (see Cianci et al (2001) Acta. Crystallographica D in press, Sept issue).
To improve throughput we would continue to develop the Optimised Anomalous Scattering (OAS) methods (Yu-Dong, et al., 1999), also referred to as Single wavelength Anomalous Dispersion (SAD).