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Introduction

Microcrystallography refers to the ability to obtain useful structures from small (< 30 μm) crystals, or small regions of “normal-sized” crystals that have bad growth habits or suffer from rapid radiation damage. This area of research is driven by scientists working on membrane proteins, for which crystal size is a major problem, or on proteins with needle-like growth habits. Because protein specimens may be hard to obtain, there is a trend toward trying to collect data from ultra-small volumes in automated screening. The presence of conformational variability, association with lipid bilayers, and a tendency towards aggregation are additional factors leading to smaller protein crystals. Successful microcrystallography involves far more than generating small X-ray beams. Here at MacCHESS we are developing the necessary capillary microbeam technology to achieve stable, small-size X-ray beams, methodologies for crystal handling and visualization, stray background reduction, specialized new detectors, and data collection protocols, all optimized for microcrystals. These novel methodologies are applied to membrane proteins that are involved in signalling responses, ion channels that are central to the establishment of membrane potentials, amyloid proteins, etc. The methods are also available to users with problematic crystals.

MacCHESS is currently able to provide microbeams through the use of focusing glass capillaries. The capillaries are produced using a custom puller, and can be drawn to a wide range of specifications. With the wiggler source, a 20 m beam with 2 mrad divergence is readily obtained, and beams as small as 5 m diameter can be produced, although the larger divergence in the latter case limits the useful crystal-detector distance. With the less-divergent undulator source, this limitation should be reduced, and we will develop capillaries optimized for the new source.

 

Experimentals

Microbeam is available at A1 and F1 MacCHESS beamlines. Capillaries are mounted in a holder which also contains a collimator, and recent improvements to the holder have made switching optics quick and easy. The current - most popular - model of capillary provides a beam of  < 20 microns at the sample.


capillary_barrel_mount

 

Highlights

N2versusHelium_KeGroup

 

Ferguson_partofneedle_plusdiffraction

Only one end of this crystal gives single lattice (Ferguson group, UPenn)

Krasnoff_small_peptideAB

A cyclic Heptapeptide from Metarhizium anisopliae (Stuart Krasnoff, USDA-ARS)

References:

Serinocylins A and B, Cyclic Heptapeptides from Metarhizium anisopliae. Stuart B. Krasnoff, Ivan Keresztes, Richard E. Gillilan, Doletha M.E. Szebenyi, Bruno G.G. Donzelli, Alice C.L. Churchill, Donna M. Gibson.Journal of Natural Products, Volume 70, Number 12, Pages 1919-1924, 2007

More information about capillaries can be found at: http://capillary.chess.cornell.edu/