Neutron Biomacromolecular Crystallography in Grenoble

M. Budayova-Spano^1/2, F. Meilleur², M. P. Blakeley¹, F. Dauvergne¹ and P. Timmins²

¹EMBL / ²ILL, 6 rue Jules Horowitz, BP 181, CEDEX 9, Grenoble, 38042, France

X-ray and neutron crystallography are highly complementary techniques. While the non-hydrogen atom positions of biomacromolecules are accurately determined by X-ray crystallography, information on the hydrogen atom positions is only possible when ultra-high resolution data (better than 1Å) can be collected. Neutron crystallography, on the other hand, can often reveal critically important hydrogen’s even at medium resolutions of 1.5 – 2.5Å. Thus neutron crystallography can provide key insight into hydrogen bond networks, water orientations and proton shuttles involved for instance in enzyme catalytic mechanisms.

Whereas development of third generation synchrotron sources has allowed X-ray protein structures to be solved from crystals of a few microns in their smallest dimensions, a major hurdle to neutron protein crystallography is that unusually voluminous crystals (~1mm³) are required to compensate for the weak flux of available neutron sources [1]. However if the protein is fully perdeuterated (all hydrogen replaced by deuterium) the required crystal volume is considerably reduced as perdeuteration largely avoids the large incoherent scattering background of hydrogen, and thus can provide a tenfold gain in the signal/noise ratio of diffraction images [2]. Obtaining large deuterated crystals suitable for neutron analysis involves performing systematic studies of the solubility and protein-protein interactions in deuterated crystallization conditions, as H-D exchange alters the physico-chemical properties of protein solutions and affects the crystallization process in a significant way [3].

We will discuss recent technological advances achieved at the ILL/EMBL Deuteration Laboratory in Grenoble in preparing D-labelled biological material, and in growing large D-labelled crystals suitable for neutron diffraction. Finally we will present one of the recent neutron protein structures determined with the Laue diffractomer (LADI) at the ILL focusing on the complex of recombinant urate oxidase enzyme with 8-azaxanthin [4].

[1] C. Bon, M. S. Lehmann, C. Wilkinson (1999), Acta Crystallogr D 55, 978-987.

[2] F. Shu, V. Ramakrishnan, B. P. Schoenborn, (2000), Proc. Natl. Acad. Sci. USA 97, 3872-3877.

[3] M. Budayova-Spano, S. Lafont, J. P. Astier, C. Ebel, S. Veesler (2000), J. Cryst. Growth 217, 311-319.

[4] M. Budayova-Spano, F. Bonneté, N. Ferté, M. El Hajji, F. Meilleur, M. P. Blakeley, B. Castro (2006), Acta Crystallogr. F 62, 306-309.