Preparing a Useful Derivative
Safety
Uranium is radioactive
Other compounds are toxic.  
 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. See the safety guide written by Dan Anderson for the use of heavy atoms.

Here is a Standard Operating Procedure (SOP) for mercuric acetate, specific to the Eisenberg lab

Wear gloves!
 
Selection
The Periodic Table
Selection: 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). 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.
Selection:
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. Usually class B metal ions are more reactive at high pH. For more details see the useful pH range list at the

Availability: There is a list of heavy atoms currently stocked in the Eisenberg Lab updated by Dan Anderson.

Table of common heavy-atom derivatizing reagents

The table below lists the most commonly cited heavy-atom derivatizing reagents as compiled from Macromolecular Structures for 1991-1994, based on Table 1 in:

Rould, M.A. "Screening for Heavy-Atom Derivatives and Obtaining Accurate Isomorphous Differences" Methods in Enzymology, Volume 276, Part A pg.465) Perpared in part by Sylvie Doublie'.

Heavy-atom Reagent

Highest stock concentration used

Soak Time

Citations

K2PtCl4

6mM

10 days

73

KAu(CN)2

20mM

-

29

Hg(CH3COO)2

50mM

-

29

Pt(NH3)2Cl2

-

-

26

HgCl2

20mM

2 days

25

K3UO2F5

-

-

23

Ethyl mercurithiosalicylate (Thimerosal)

0.8mM

10-30 days

22

(K/Na)AuCl4

-

-

22

(Na/K)3IrCl6

5mM

-

21

CH3CH2HgPO4

-

-

20

K2PtCl6

5mM

-

19

UO2(NO3)2

-

-

17

K2Pt(NO2)4

10mM

7 days

17

(CH3)3Pb(CH3COO)

-

-

14

CH3HgCl

-

-

13

p-Chloromercuribenzene sulfate (PCMBS)

10mM

-

13

K2Pt(CN)4

5mM

-

12

Di-m-iodobis(ethylenediamine) diplatinum (PIP)

-

-

12

Pb(CH3COO)2

100mM

1 day

12

K2HgI4

5mM

days

12

Mersalyl

0.9mM

10-40 days

12

p-Chloromercuribenzoate (PCMB)

0.8mM

10-30 days

11

CH3Hg(CH3COO)

-

-

11

C(HgOOCH3)4 Tetrakis(mercuriacetoxy)methane (TAMM)

-

-

10

SmCl3

20mM

-

8

K2OsO4

-

-

8

(K/Na)2OsCl6

-

-

7

1,2-Diacetoxymercuri- 2,3-dimethoxybutane (Baker's dimercurial)

10x Protein concentration

-

6

2-Chloromercuri-4-nitrophenol

-

-

6

AgNO3

-

-

5

CH3CH2HgCl

Saturated

4 days

5

p-Hydroxymercuribenzoate

-

-

5



Screening
from Bogon & Shapiro, 2000  
 Screening: Screening for heavy atom derivatives has never been easier! Thanks to the observation by Bogon & 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

  • It has saved much time for students at UCLA. It can work for you!

    See T. J. Boggon and L. Shapiro Structure 8, R143-R149, 2000.

  •  Soaking: Once you have identified the heavy atom conditions that produce a native gel band shift, you can try soaking the heavy atom into your crystal.
    Procedure for soaking heavy-atom reagents into macromolecular crystals:
    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.


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