| We propose developing methods for structure determination of three-dimensional nano-crystals of extremely radiation sensitive molecules, like proteins and organic pharmaceuticals. If protein crystals have multiple layers, but are smaller than 1 m, they are currently beyond the reach of crystallographic structure determination, whether by X-rays or electrons. This has prompted concerted (multi-million Euro) international projects for building ever more focussed, brilliant synchrotron X-ray beamlines for crystals in the micrometer range. However, for structure determination of 3D protein crystals that are smaller than about 0.5 μm, it can be shown that electrons are be much more suited for structure determination than X-rays, as they are less damaging by several orders of magnitude when normalised to the amount of elastically scattered quanta. Indeed, if only two-dimensional, single-layer crystals of proteins are available, electron diffraction already is the method of choice. However, if such crystals have multiple layers, practical problems are the data acquisition, the lack of software to process such data and the absence of successful pilot studies. These drawbacks currently prompt most protein crystallographers to put their efforts into growing larger crystals that diffract X-rays, and make them abandon projects if such crystals cannot be obtained. In order to demonstrate that electron crystallography is a good alternative for sub-micron 3D protein crystals, we propose: - developing more robust, standard (automated) protocols for sample handling and data acquisition; - developing/improving data processing procedures for unit cell determination and indexing of electron diffraction patterns; - adapting standard X-ray data analysis software for subsequent steps in structure determination; - exploring existing and novel concepts in phasing electron diffraction data, treating dynamic scatter and refining structures against electron diffraction data; - validating these concepts in practical structure determination of various (membrane) proteins and (pharmaceutical) organic compounds with known and unknown structures. In all approaches, we will implement multi-variate, maximum likelihood statistical methods. The Delft group is one of the pioneering groups in electron crystallography of radiation hard materials, the Leiden group has a track record of protein structure determination and methods development in X-ray crystallography. This proposal builds on a Leiden-Delft collaboration in which we have shown that it is possible to reproducibly grow protein nano-crystals. We developed sample preparation procedures and data collection protocols that allow us to collect electron diffraction data of 3D protein crystals to a resolution of 2 Å, and of pharmaceuticals to 0.8 Å. Because of the high radiation sensitivity of these crystals, it is not possible to align them in the electron beam prior to data collection, hence only single diffraction shots of randomly oriented crystals can be obtained. This is similar to data collection procedures in X-ray crystallography of weakly diffracting protein crystals that cannot be frozen, like, for instance, crystals of infectious viruses. We showed that the unit cell parameters can be deduced by analysing data sets of random electron diffraction patterns. We developed a computer program for unit cell determination which we will start distributing at the end of 2008. Initial publications of our work on protein nano-crystallography by electron diffraction resulted in invitated talks at the ACA 2008 in Knoxville, the IUCr 2008 in Osaka, the EMC 2008 in Aachen and an invited plenary lecture at the ECM 2009 in Istanbul. The Leiden group is a member of the CERN-directed Medipix consortium, which is developing a quantum area detector for high energy radiation. |
