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Fluorescent Approaches to High Throughput CrystallographyX-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1 %, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. Preliminary experiments show that the presence of the fluorescent probe does not affect the nucleation process or the quality of the X-ray data obtained.
Document ID
20050207550
Acquisition Source
Marshall Space Flight Center
Document Type
Conference Paper
Authors
Pusey, Marc L.
(NASA Marshall Space Flight Center Huntsville, AL, United States)
Forsythe, Elizabeth
(BAE Systems Huntsville, AL, United States)
Achari, Amiruddha
(Raytheon Co. Huntsville, AL, United States)
Date Acquired
August 23, 2013
Publication Date
January 1, 2005
Subject Category
Solid-State Physics
Meeting Information
Meeting: American Cystallographic Association Conference
Location: Orlando, FL
Country: United States
Start Date: May 27, 2005
End Date: June 2, 2005
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

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