Research Article: In situ macromolecular crystallography using microbeams

Date Published: May 01, 2012

Publisher: International Union of Crystallography

Author(s): Danny Axford, Robin L. Owen, Jun Aishima, James Foadi, Ann W. Morgan, James I. Robinson, Joanne E. Nettleship, Raymond J. Owens, Isabel Moraes, Elizabeth E. Fry, Jonathan M. Grimes, Karl Harlos, Abhay Kotecha, Jingshan Ren, Geoff Sutton, Thomas S. Walter, David I. Stuart, Gwyndaf Evans.

http://doi.org/10.1107/S0907444912006749

Abstract

A sample environment for mounting crystallization trays has been developed on the microfocus beamline I24 at Diamond Light Source. The technical developments and several case studies are described.

Partial Text

The mounting of crystals in loops or on meshes and their cryoprotection remains a manual and often painstaking process in macromolecular crystallography (MX), in contrast to the successful automation of many other steps in the sequence-to-structure pipeline. Some years ago, the measurement of diffraction data from crystals at room temperature within the crystallization trays used for their growth was shown to be feasible on a bending-magnet beamline (Jacquamet et al., 2004 ▸). Jacquamet and coworkers demonstrated that a large defocused X-ray beam of ∼1 mm in size could be used to provide useful information about the diffraction quality of crystals within a drop. In certain cases, diffraction data sets could also be measured from crystals of a few hundred micrometres in size. More recently, ligand-soak experiments have been performed in situ (le Maire et al., 2011 ▸), and at the Swiss Light Source a crystallization facility now adjoins a beamline capable of in situ data collection (Bingel-Erlenmeyer et al., 2011 ▸). These implementations use a robotic arm to support and manipulate crystallization plates in order to perform measurements. The positional resolution and repeatability of these robotic arms limit their usefulness for crystal and X-ray beam sizes much less than 20 µm, sizes that are typical for a microfocus beamline such as I24.

The ability to obtain information on the diffraction quality of crystallization hits in situ has proved particularly valuable in overcoming the challenges of membrane-protein crystallization and cocrystallizations to produce complexes, in which the effective search space for optimum conditions is greatly increased with each additional component. Indeed, in situ characterization is now a routine part of the optimization pipeline for the MPL, with regular access on a roughly monthly basis subject to the operations schedule as part of the beamline user time. To date, it has been applied to over a dozen MPL projects. This method of data collection typically allows the experimenter to record improved diffraction data, reduces the risk of false negatives, enables a faster progression of crystallization optimization and eliminates crystal manipulation. What the early experiments have shown strikingly is that in addition to this role in screening crystallization hits, in situ data collection can yield data sets of high quality and may offer an attractive path to routine structure determination for certain potentially challenging structures.

 

Source:

http://doi.org/10.1107/S0907444912006749

 

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