Serial crystallography using synchrotron radiation - novel strategies for microcrystallography

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.

EMBL beamlines for macromolecular crystallography at PETRA III

Since 2012, EMBL Hamburg operates two new beamlines for macromolecular crystallography - P13 and P14 - at PETRA III at DESY (Hamburg, Germany). We exploit the high brilliance and the wide energy range offered by PETRA III to offer a wide range of conditions to fit the experimental conditions to the challenges posed by the samples. P13 provides high photon flux down to 4 keV. With a helium cone and a kappa goniostat, this allows optimized data collection for SAD phasing. Using adaptive mirrors, the focus size (H x V) can be adjusted between 30 x 20 μm^2 and 150 x 100 μm^2 to match the size of the sample. A MARVIN sample changer is in operation for rapid loading and unloading of samples. P14 offers a high photon flux (>10^12 ph/sec at 12 keV into 5 x 5 µm^2). The beamsize can be varied between 1 x 1.5 mm^2 (unfocused) and 5 x 5 µm^2 (fully focused) in less than a minute by moving the KB mirrors in and out of the beam. For small crystals, an MD3 vertical diffractometer with a sphere of confusion smaller than 100 nm offers excellent conditions. Both beamlines are equipped with PILATUS 6M-F detectors for shutter-less data collection and dedicated data processing computers. The beamlines are embedded into the 'Integrated Facility for Structural Biology' offering facilities for sample preparation and characterization, a laboratory specifically equipped for the preparation of heavy atom derivatives, and downstream facilities for data evaluation We will report about the status of the beamlines and describe typical experimental situations (small crystals, large unit cells, serial crystallography, low-energy phasing, small molecules and others).

Serial femtosecond X-ray diffraction of in vivo crystals in intact yeast cells

Peroxisomes are membrane-enclosed organelles in eukaryotic cells with important roles in lipid metabolism and the scavenging of reactive oxygen species. Peroxisomes are capable of carrying an unusually high load of proteins, which under appropriate nutrient conditions results in the in situ crystallization of peroxisomal proteins in several yeast species and vertebrate hepatocytes [1,2]. In the methylotrophic yeast H.polymorpha, the predominant peroxisomal protein alcohol/methanol oxidase (AO) oligomerizes into octameric assemblies with a molecular mass of 600 kDa that spontaneously form 200-500 nm crystallites within peroxisomes [1]. We exposed H.polymorpha cell suspensions containing peroxisome-confined AO crystallites to femtosecond X-ray pulses at the Coherent X-ray Imaging (CXI) experimental endstation at the Linac Coherent Light Source. Peak detection routines mining the resulting scattering profiles identified >5000 Bragg-sampled diffraction patterns, providing the proof of concept that background scattering from the cells does not deteriorate the signal-to-noise ratio to an extent precluding observation of diffraction from individual AO crystallites. Summation patterns assembled from the individual frames match low-resolution powder diffraction patterns from concentrated suspensions of purified peroxisomes collected at the P14 beamline at the PETRAIII synchrotron, confirming that the observed diffraction mainly results from Bragg scattering of peroxisomal crystallites. To the best of our knowledge our data are the first to report room temperature X-ray diffraction from functional protein crystals in their native cellular environment. Currently the maximum resolution achieved in the diffraction patterns is limited to 20-15 Å. Future work will need to address improved sample preparation protocols in order to assess whether diffraction to a resolution sufficient to permit structure solution can be obtained. Protein crystal formation in vivo has been observed under physiological or pathological conditions in a number of other systems [3]. We hope that our results will help to establish serial femtosecond X-ray diffraction (SFX) as a method for structural characterization of cellular structures with crystalline content and provide a proof of concept for using in situ crystallization of proteins as a means to generate nanocrystalline samples for SFX.