Many proteins do not form three-dimensional (3D) crystals large enough and of sufficient quality for their structure to be determined by X-ray crystallography. This project focuses on the use of 3D nanocrystals and electron crystallography. A single protein nanocrystal is continuously rotated in the electron beam to obtain a complete electron diffraction data set. The total exposure dose must be low to minimize sample damage, which means that the detector must be extremely sensitive.
There are several way to obtain structural information from such sub-micron sized 3D crystals: employ intense XFEL sources, electron imaging and electron diffraction. Solving new structures using these approaches remains a problem, as ab-initio crystal phasing is hampered by experimental constraints that severely compromise data quality. These constraints include incomplete sampling of Bragg spots and (for electron diffraction), dynamic and inelastic scattering.
Complete sampling of Bragg spacings can be obtained using a fast Timepix quantum area detector and continuously rotating the nanocrystal in the beam. As this detector is of unprecedented sensitivity, the electron diffraction data can be collected under low dose conditions.
A rotation series of 40 degrees collected with very fine slicing from a single frozen 200-nm thick lysozyme nanocrystal showed diffraction up to 2.0 Å resolution.
Furthermore, for the beam sensitive pharmaceutical compound carbamazepine, a complete dataset was collected from a flat 200-nm thick nanocrystal at room temperature using a total exposure dose of no more than 4.0 e-/Å2. The collected diffraction patterns showed high-resolution Bragg spots up to 0.8 Å.