High-resolution protein structures determined by X-ray diffraction require the production of high-quality, well-ordered crystals. The generation of such crystals typically involves the identification of initial crystallization conditions, followedby optimization to produce well-ordered crystals, and subsequent X-ray diffraction, most commonly performed at synchrotron facilities. Each step in this pipeline can typically require weeks or months to assess success or failure in iterative optimizations, limited by a combination of slow kinetics for protein nucleation and growth in order to generate large well-ordered single crystals, and limited access to synchrotron facilities for assessing crystal quality and performing data collection. We propose the use of nonlinear optical imaging to rapidly inform the final three key experimental steps of initial crystal formation, optimization of crystal quality prior to diffractin, and synchrotron X-ray diffraction analysis. For the first goal, intercalation of protein crystals wth SHG-phores is proposed for enhancing the range and sizes of crystals detectable by SHG, with proof-of-concept measurements suggesting enhancement factors of 1000-fold are achievable. In situ assessment of crystal quality is proposed for rapid optimization based the use of polarization-dependent SHG imaging for identification of multi-domain, twinned, and highly mosaic crystals. Finally, instrumentation to enable rapid serial crystallography of micro- and nano-crystalline showers are proposed based on integration of synchrotron XRD with multi-modal confocal reflectance, brightfield transmittance, birefringence, two-photon excited ultraviolet fluorescence (TPE-UVF), and UV-SHG, all acquired simultaneously at up to video rate and with perfect image registry. Using this combined measurement suite, we aim to enable routine and confident detection of crystals 1-5mm in diameter to enable diffraction measurements with a 1 mm collimated source. The proposed efforts build on a foundation laid during the previous initial cycle of NIH support. SHG and TPE-UVF microscopy first proposed in our previous period of support are now established and widely used methods within the crystallography community. Previous support directly contributed to 35 peer-reviewed journal articles, 66 presentations, and 3 issued patents. Our technology has been licensed, and a commercial SHG/TPE-UVF microscope is now available for protein crystal screening. In addition, a custom first-generation nonlinear optical imaging instrument has been integrated into a macromolecular crystallography beamline at Argonne National Laboratory.
Nonlinear optical imaging will be used to rapidly close key feedback loops within the structural biology pipeline. The unique symmetry properties of second harmonic generation (SHG) result in no coherent signal production from disordered aggregated or solvated protein, but highly selective signal from the large majority of space groups in which proteins can crystallize. It is proposed that relationships between the detected SHG images and the presence, quality, and position of protein crystals can greatly reduce the overall time required for structure determination.
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