Long-wavelength Mesh&Collect native SAD phasing from microcrystals.

Michele Cianci,Max Nanao,Thomas R Schneider

doi:10.1107/s2059798319002031

Abstract

Harnessing the anomalous signal from macromolecular crystals with volumes of less than 10 000 µm3 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 ∼20 µm or less using an accordingly sized microbeam at a wavelength of 1.892 Å (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 Mn2+ and Ca2+ 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° 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.

Highlights

De novo determination of macromolecular structures requires the accurate measurement of structure factors and retrieval of experimental phases from the crystals of the given specimen
On the EMBL beamline P13 at PETRA III, Mesh&Collect data collection from concanavalin A microcrystals with linear dimensions of $20 mm or less using an 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), allowing experimental phase determination using the anomalous signal from naturally present Mn2+ and Ca2+ ions
We show here that when long wavelengths (Djinovic Carugo et al, 2005) are combined with the Mesh&Collect datacollection approach (Zander et al, 2015) and when a genetic algorithm (GA) is used to compile a data set (Zander et al, 2016), a native SAD (Rose et al, 2015) experiment can yield a structure solution from microcrystals

Summary

Introduction

De novo determination of macromolecular structures requires the accurate measurement of structure factors and retrieval of experimental phases from the crystals of the given specimen. When a model with significant structure similarities is available, phases can be retrieved using the molecular-replacement (MR) method. One experimental phasing method that is gaining popularity is the use of the anomalous signal from naturally occurring anomalous (native SAD) or from ad hoc incorporated anomalous scatterers (International Tables for Crystallography, 2012). Along with the growing popularity of anomalous scattering for phasing, X-ray radiation damage became a general concern for any data collection performed on an undulator beamline, resulting in systematic analyses of synchrotron data sets at room temperature or cryotemperatures, with samples showing the characteristic ‘fingerprints’ of radiation damage (Helliwell, 1988; Ravelli & McSweeney, 2000; Borek et al, 2007).

Methods

Results

Conclusion