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).

Monoclinic HEWL derivative structure at 0.75 Å resolution

Tris-dipicolinate lanthanide complex, Na₃.[Ln(DPA)³], where DPA stands for pyridine-2,6 dicarboxylate, has been proven to be especially interesting to produce derivative crystals with high phasing power [1]. The lanthanide complex can act as a cross-linking agent, which binds at the interface between protein molecules, leading to a supramolecular interaction at the crystal scale [2]. In the case of hen egg-white lysozyme (HEWL), derivative crystals obtained by co-crystallization with Yb(DPA)₃^3- belong to the space group C2, in similar crystallization conditions that normally lead to the tetragonal form P4₃2₁2. Data were collected on a derivative crystal up to a resolution of 0.75 Å at the EMBL beamline P13 at PETRA III at DESY (Hamburg, Germany). Taking advantage of the high the resolution and the strong anomalous signal of the Yb³⁺ (f´´ = 5.2 e^- at 17 keV), the structure was solved by both SAD and ab initio methods. Data collection, experimentally phased electron density maps and the structure, especially with respect to the vicinity of the lanthanide binding sites, will be discussed.