Preparing Heavy Metal Derivatives
Safety: Heavy atoms are toxic to humans, and uranyl compounds are radioactive. When preparing heavy atom derivatives, special care must be taken to prevent poisoning/irradiating yourself or others.
Handling precautions: Wear gloves when handling heavy-atoms and avoid direct contact with the skin. If contact occurs, wash the contaminated area thoroughly with soap and water. Promptly clean up spills.
Read the complete heavy metal handling, safety, and disposal protocol before continuing.
Choosing the right heavy metal:
- Cystein, hisitidine, and methionine are reactive with Class B heavy atoms (Hg, Au, Pt, Ir, etc.)
- Glutamic acid and Aspartic acid are reactive toward Class A heavy atoms (Lanthanides, Actinides, e.g. U, Sm)
- Lanthanides and Actinides tend to have the strongest anomalous signal. To check how many anomalous electrons are in a given element see Ethan Merritt's X-ray Anomalous Scattering Tables
- One should also consider how heavy atom reactivity varies with pH
- A concise review is given by Petsko in: Petsko, G.A., "Perparation of Isomorphous Heavy-Atom Derivatives Methods in Enzymology, Volume 114, , pages 147-157. and more exhaustively in: Blundel & Johnson's book, "Protein Crystallography" pages 183-239
- Recipes for iodide and cesium derivatives
- Phasing with potassium iodide- a powerpoint presentation
- Recipe for selenomethionyl derivatives
Table of common heavy-atom derivatizing reagents (from "Screening for heavy-atom derivatives and obtaining accurate isomorphous differences," M.A. Rould, Methods in Enzymology, 276, pp 461-72 (1997).
Screening:
Screening for heavy atom derivatives has never been easier! Thanks to the observation by Boggon & Shapiro that the usefulness of a heavy atom compound can be screened by means of a simple native gel shift assay. Benefits of the method include the following features:
- does not require the use of crystals!
- quickly eliminate denaturing conditions by identifying which heavy atoms prevent the protein from entering the gel
- the assay can be performed quickly on a PHAST gel system
- you can screen dozens of heavy atom conditions in one afternoon
- false negatives are rare
- reliable: no false positives detected in this report
See T. J. Boggon and L. Shapiro Structure 8, R143-R149, 2000.
Soaking:
Soaking is aided by placing the crystal in an artificial mother liquor which has been shown to keep the crystal stable. To minimize the volumes of heavy-atom reagent that will be added to the crystal mother liquor (less than 10% of original droplet volume), relatively high stock concentrations (20 to 50mM or higher) of the heavy atom compounds will have to be made. With the crystal sitting in ~10ul of the artificial mother liquor, add ~0.2ul of the heavy-atom solution with a long, thin gel-loading-pipet-tip away from the crystal. Watch the crystal under a microscope for a few minutes looking for any telltale signs of crystal cracking, loss of birefringence or melting. Note if the solution remains clear especially where the heavy-atom was added. If nothing happens within 30-60 minutes add another aliquot of heavy-atom solution and continue this process until the final heavy-atom concentration is between 2 to 5mM. Let the crystal sit for an appropriate amount of time, typically between four hours and three days. The crystal may or may not change color depending upon the compound used. To reduce non-specific binding it may be good idea to back-soak the crystal for two to six hours in heavy-atom free mother liquor just prior to diffraction analysis.
Adapted from UCLA