Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4 A resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hair's cuticles, explaining the strong nucleating activity of the surface.

An algorithm is described that calculates the most likely primitive unit cell given a set of randomly oriented electron-diffraction patterns with unknown angular relationships.

The drive for elucidation of important macromolecular structures to high resolution in their 3D native or near-native state places continuously higher demands on the quality of the experimental data. For instance, recording of diffraction patterns good enough for structural studies from cryo-preserved bio-macromolecules at low dose conditions remains challenging and highly desirable. The emergence of hybrid pixel detectors opens up new possibilities for direct electron detection and superior detector performance. Here, we report on the characteristics of the Medipix2 detector in diffraction studies, with a special focus on the reliability of the intensities acquired in very low dose conditions. Diffraction data recorded on a Medipix2 detector were assessed in refinement analysis. R-factors lower than 10% were obtained from data recorded at electron dose of 0.05 el/Å 2 . The reproducibility of the data was also shown to be high, given the correlation coefficient of the intensities being higher than 0.9970. The contrast that could be achieved at very low dose conditions was at least an order of magnitude better than that of image plates, based on a direct comparison.

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