Abstract
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼10(3)-10(4)-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.
Highlights
Diffraction-quality protein crystals are typically obtained through crystallization screenings, followed by optimization, and are placed into cryoloops, which are flash-cooled in liquid nitrogen to reduce X-ray damage and aid in sample handling (Dobrianov et al, 1999; Karain et al, 2002)
From the resulting X-ray diffraction images recorded as a function of sample position in the beam, protein crystals are centered based on the locations of strongest Bragg-like diffraction
Both the presence and position of the crystal can be independently confirmed with bright-field imaging (a), Nonlinear optical (NLO) microscopy and X-ray diffraction (XRD) measurements
Summary
The high photon flux and energy tunability of synchrotron radiation sources have made them indispensable tools for X-ray analysis, with applications spanning protein structure determination through materials science and nanotechnology (Rasmussen et al, 2011; Moukhametzianov et al, 2008; Bates et al, 2006; Berger et al, 2010; Dauter, 2006; Ihee et al, 2010; le Maire et al, 2011; Parker et al, 2006; Riekel et al, 2005). The increasing drive toward tighter focusing has enabled structure determination on ever-smaller crystals and subdomains within materials, but presents growing challenges for reliable crystal centering. These challenges are relevant for protein crystal diffraction, in which the drive. 20, 531–540 toward fully automated X-ray diffraction analysis at synchrotron sources has introduced bottlenecks in sample positioning (Andrey et al, 2004; Moukhametzianov et al, 2008; Pothineni et al, 2006; Aishima et al, 2010; Cherezov et al, 2009; Stepanov et al, 2011a). X-ray fluorescence raster is relatively fast, but it requires a convenient X-ray fluorescent element to be present in the crystal (Stepanov et al, 2011a)
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