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abstracts
Protein crystallography continues to be one of the most frequently used techniques to obtain structural information of biomacromolecules to atomic resolution. Since protein crystals of delicate target systems are often limited in size, one of the main goals in the design of modern beamlines is the construction of highly intense X-ray beams with small focal size to obtain high resolution diffraction images of microcrystals. However, this development has led to the situation, that the full intensity of the beam can destroy a protein crystal within fractions of a second. Therefore often only a small number of diffraction patterns can be obtained from one single crystal. Here we describe the adaptation of the serial crystallography approach, which has first been developed at X-ray Free-Electron Lasers (Chapman et al. 2011) to the usage of a microfocus synchrotron beamline, using a standard cryogenic loop for sample delivery. We proved this concept with in vivo grown cathepsinB microcrystals (TbCatB, Koopmann et al. 2012, Redecke et al. 2013) (average of 9 μm3), a medically and pharmaceutically relevant protein, involved in the life cycle of T. brucei. In these experiments it was possible to show that serial crystallography enables the utilization and outcome of the above described bottlenecks and features of modern 3rd generation synchrotron microfocus beamlines. Our strategy exploits the combination of a micron-sized X-ray beam, high precision diffractometry and shutterless data acquisition with a pixel-array detector. By combining the data of 80 TbCatB crystals, it was possible to assemble a dataset to 3.0 Å resolution. The data allow the refinement of a structural model that is consistent with that previously obtained using FEL radiation, providing mutual validation.
abstracts
Spontaneous protein crystallization within living cells has been observed several times in nature, e.g. for storage proteins in seeds. In vivo crystal growth can also occur during gene over-expression, as particularly discovered in baculovirus-infected insect cells [1]. We have recently shown that these in vivo crystals represent valuable targets for structural biology after isolation from the cell. Applying serial crystallography techniques at an X-ray free-electron laser (XFEL) as well as using a highly brilliant synchrotron source, single crystal diffraction pattern were collected and combined to yield high-resolution structural information of the associated fully glycosylated protein [2,3]. So far, the cellular mechanisms involved in the in vivo crystallization process remain to be understood, preventing a more successful application of this novel approach. Thus, our study aims at identifying the parameters crucial for optimal crystal growth within baculovirus-infected Sf9 insect cells. Combining confocal microscopy with live-cell imaging techniques and compartment-specific staining methods, we systematically investigated the impact of the intracellular environment on in vivo crystallization by directing recombinant proteins into different cellular compartments using specific signal sequences. Moreover, the impact of cellular transport mechanisms and induced cellular stress on the quality and size of the in vivo crystals was investigated in detail. The presented results provide important insights into the process of protein crystallization within living cells and will therefore significantly contribute to increase the success rate for spontaneous crystal growth of other proteins. Considering that in vivo crystals represent highly suitable targets for structural biology, this approach offers exciting new possibilities for proteins that do not form crystals suitable for conventional X-ray diffraction in vitro.
