Abstract
Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II—AMX and FMX—deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins—Akt1, PI3Kα, and CDP-Chase—to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or “raster-collect”, a more automated approach for a larger number of crystals (using CDP-Chase as an example).
Highlights
Macromolecular X-ray crystallography has revolutionized the study of proteins, providing atomic level detail that offers insight into fundamental molecular mechanisms and guides drug design.Historically, the rate-limiting step in structure determination has been the growth of crystals of sufficient size and homogeneity to yield high quality X-ray diffraction
A vector was defined from one end of the crystal to the other andAkt1
PIP3 recruits the protein kinase Akt1 to the plasma membrane, which is fully activated upon phosphorylation of its activation loop by PDK1 and
Summary
The rate-limiting step in structure determination has been the growth of crystals of sufficient size and homogeneity to yield high quality X-ray diffraction. Extensive screening and optimization are undertaken, but often fail to yield large, well-diffracting crystals. Molecules 2019, 24, 496 advent of third generation synchrotrons and microfocus beams, the need for such time-consuming optimization is rapidly decreasing [1]. Small (
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