Abstract
This paper recounts the first successful cryo-cooling of protein crystals that demonstrated the reduction in X-ray damage to macromolecular crystals. The project was suggested by David C. Phillips in 1965 at the Royal Institution of Great Britain and continued in 1967 at the Weizmann Institute of Science, where the first cryo-cooling experiments were performed on lysozyme crystals, and was completed in 1969 at Purdue University on lactate dehydrogenase crystals. A 1970 publication in Acta Crystallographica described the cryo-procedures, the use of cryo-protectants to prevent ice formation, the importance of fast, isotropic cryo-cooling and the collection of analytical data showing more than a tenfold decrease in radiation damage in cryo-cooled lactate dehydrogenase crystals. This was the first demonstration of any method that reduced radiation damage in protein crystals, which provided crystallographers with suitable means to employ synchrotron X-ray sources for protein-crystal analysis. Today, fifty years later, more than 90% of the crystal structures deposited in the Protein Data Bank have been cryo-cooled.
Highlights
Introduction to prolong the usefulX-ray life of frozen . . . protein crystalsThese words in the last paragraph of the April 1968 paper describing the first successful protein-crystal cryo-cooling experiments proved to be prophetic (Haas, 1968b).During the 1990s, the cryo-cooling of protein crystals became a universal technique for reducing the X-ray damage from synchrotron X-ray sources
In April 1967, hen eggwhite lysozyme crystals were successfully cryo-cooled in the Crystallography Department at the Weizmann Institute of Science, where I continued a project that began at the Royal Institution of Great Britain in 1965
Employing a simple cold gas stream (198 K) with a dry nitrogen coaxial stream from a single Dewar of liquid nitrogen, native lysozyme crystals with a sucrose cryo-protectant were exposed to the primary X-ray beam from a standard sealed Philips X-ray tube for several days without showing any noticeable radiation damage
Summary
After my graduate work with David Harker (Tulinsky, 1996) and graduating from the University of Buffalo in February 1965 (Haas, 1965), my wife and I traveled to London in September 1965 for postdoctoral work at the Royal Institution of Great Britain. Lysozyme crystals could be cross-linked with glutaraldehyde to make them more robust (Quiocho & Richards, 1964), and this may reduce the X-ray damage This bireactant aldehyde connects nearby lysine side chains on the protein surface (intermolecularly and intramolecularly) and forms a single crystal consisting of millions of lysozyme molecules covalently bound together. Tantly, with denatured cross-linked lysozyme crystals (swelled, both fully and surface cross-linked), slowly removing the denaturant and returning the crystal back to its original supernatant caused the ‘gel’ crystal to shrink again and recover its X-ray diffraction pattern (Fig. 3) This proved to be a remarkable renaturation property which shows that the protein molecules can rearrange/recrystallize themselves. I worked alone on this project for the entire time at the Royal Institution
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