Strategies for experimental phasing at P13
by Pompidor Guillaume (EMBL)
at 25b ( 109 )
The P13 Macromolecular Crystallography beamline, operated by the EMBL at Petra III, enables diffraction data collection over a wide energy range, from 4 keV to 17.5 keV (0.7 – 3.1 Å). In order to fully exploit the beamline capability at low energy (1012 ph.s-1 at 5 keV), the PILATUS 6M-F detector, with a 450 μm Si sensor thickness and with a custom calibration at low energy, is mounted on a 2 θ-angle stage to increase the maximum resolution achievable (2.4 Å at 4keV).
We will present and describe the typical experimental setup used for limiting the air absorption (He-cone mounted on the detector) for long wavelength data collection, as well as several examples of structure determination using intrinsic anomalous scatterers, i-e sulfur SAD or calcium (MAD at the Ca K edge at 4.05 keV).
In a second part, we will present a new tool for structure determination using an engineered nanobody. A generally applicable lanthanide-binding motif has been engineered onto the nanobody fold, resulting in constructs displaying nanomolar affinity for terbium(III) and unmodified antigen binding characteristics. As a proof of concept, we will present the structure the eGFP (enhanced Green Fluorescent Protein) in complex with the engineered nanobody and discuss a general application of the method.