Synchrotron radiation diffraction from two-dimensional protein crystals at the air/water interface.

Protein structure determination by classical x-ray crystallography requires three-dimensional crystals that are difficult to obtain for most proteins and especially for membrane proteins. An alternative is to grow two-dimensional (2D) crystals by adsorbing proteins to ligand-lipid monolayers at the surface of water. This confined geometry requires only small amounts of material and offers numerous advantages: self-assembly and ordering over micrometer scales is easier to obtain in two dimensions; although fully hydrated, the crystals are sufficiently rigid to be investigated by various techniques, such as electron crystallography or micromechanical measurements. Here we report structural studies, using grazing incidence synchrotron x-ray diffraction, of three different 2D protein crystals at the air-water interface, namely streptavidine, annexin V, and the transcription factor HupR. Using a set-up of high angular resolution, we observe narrow Bragg reflections showing long-range crystalline order in two dimensions. In the case of streptavidin the angular range of the observed diffraction corresponds to a resolution of 10 A in plane and 14 A normal to the plane. We show that this approach is complementary to electron crystallography but without the need for transfer of the monolayer onto a grid. Moreover, as the 2D crystals are accessible from the buffer solution, the formation and structure of protein complexes can be investigated in situ.