6h2m

Publication Abstract from PubMed

Harnessing the anomalous signal from macromolecular crystals with volumes of less than 10 000 microm(3) for native phasing requires careful experimental planning. The type of anomalous scatterers that are naturally present in the sample, such as sulfur, phosphorus and calcium, will dictate the beam energy required and determine the level of radiation sensitivity, while the crystal size will dictate the beam size and the sample-mounting technique, in turn indicating the specifications of a suitable beamline. On the EMBL beamline P13 at PETRA III, Mesh&Collect data collection from concanavalin A microcrystals with linear dimensions of approximately 20 microm or less using an accordingly sized microbeam at a wavelength of 1.892 A (6.551 keV, close to the Mn edge at 6.549 keV) increases the expected Bijvoet ratio to 2.1% from an expected 0.7% at 12.6 keV (Se K edge), thus allowing experimental phase determination using the anomalous signal from naturally present Mn(2+) and Ca(2+) ions. Dozens of crystals were harvested and flash-cryocooled in micro-meshes, rapidly screened for diffraction (less than a minute per loop) and then used for serial Mesh&Collect collection of about 298 partial data sets (10 degrees of crystal rotation per sample). The partial data sets were integrated and scaled. A genetic algorithm for combining partial data sets was used to select those to be merged into a single data set. This final data set showed high completeness, high multiplicity and sufficient anomalous signal to locate the anomalous scatterers, and provided phasing information which allowed complete auto-tracing of the polypeptide chain. To allow the complete experiment to run in less than 2 h, a practically acceptable time frame, the diffractometer and detector had to run together with limited manual intervention. The combination of several cutting-edge components allowed accurate anomalous signal to be measured from small crystals.

Long-wavelength Mesh&Collect native SAD phasing from microcrystals.,Cianci M, Nanao M, Schneider TR Acta Crystallogr D Struct Biol. 2019 Feb 1;75(Pt 2):192-199. doi:, 10.1107/S2059798319002031. Epub 2019 Feb 11. PMID:30821707^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.