Facilitating best practices in collecting anomalous scattering data for de novo structure solution at the ESRF Structural Biology Beamlines.

Daniele De Sanctis,Olof Svensson,Marcus Oscarsson,Gordon Leonard,Alexander Popov

doi:10.1107/s2059798316001042

Abstract

The constant evolution of synchrotron structural biology beamlines, the viability of screening protein crystals for a wide range of heavy-atom derivatives, the advent of efficient protein labelling and the availability of automatic data-processing and structure-solution pipelines have combined to make de novo structure solution in macromolecular crystallography a less arduous task. Nevertheless, the collection of diffraction data of sufficient quality for experimental phasing is still a difficult and crucial step. Here, some examples of good data-collection practice for projects requiring experimental phasing are presented and recent developments at the ESRF Structural Biology beamlines that have facilitated these are illustrated.

Highlights

The constantly increasing number of macromolecular crystal structures deposited in the Protein Data Bank (PDB; Berman et al, 2007), the increasing number of protein folds that these contain and the advent of more sensitive methods (McCoy et al, 2007; Read, 2001; DiMaio et al, 2011) has meant that molecular replacement (MR) is the overwhelming choice for structure solution in macromolecular crystallography (MX)
We briefly describe data collection strategies aimed at reducing both systematic errors and radiation damage during multiwavelength anomalous dispersion (MAD)/single-wavelength anomalous dispersion (SAD) experiments and describe how such strategies can best be put into practice at the ESRF Structural Biology Beamlines
Sets at different positions it is possible to increase multiplicity while escaping radiation damage; for highly radiationsensitive samples partial data sets can be collected at each position and merged to produce a single complete data set to higher resolution than might otherwise have been the case; in MAD experiments data sets at different energies can be collected from different positions of the same crystal

Summary

Introduction

The constantly increasing number of macromolecular crystal structures deposited in the Protein Data Bank (PDB; Berman et al, 2007), the increasing number of protein folds that these contain and the advent of more sensitive methods (McCoy et al, 2007; Read, 2001; DiMaio et al, 2011) has meant that molecular replacement (MR) is the overwhelming choice for structure solution in macromolecular crystallography (MX). Most experiments for de novo structure determination routinely exploit anomalous scattering via the multiwavelength anomalous dispersion (MAD; Smith, 1991; Hendrickson, 1991) or single-wavelength anomalous dispersion (SAD; Rice et al, 2000; Dauter, 2002; Dauter et al, 2002) techniques. Such experiments are facilitated by the almost continuous evolution of tunable synchrotron beamlines at which experimenters are able to accurately measure the absorption edges of almost any anomalous scatterer that can be introduced into a crystal and to collect diffraction data at various energies around these in order to optimize anomalous and dispersive signals. All three endstations share a similar data-collection geometry (! axis for oscillation scans horizontal and perpendicular to the X-ray beam) and are equipped with MK3 minikappa goniometers (Brockhauser et al, 2013) for crystal realignment and

ESRF MAD Structural Biology Beamlines

The MXCuBE2 concept

Calculation of data-collection strategies

Crystal reorientation

Multi-positional data collection

Conclusions