Standard Operating Procedure
for Derivatization of Macromolecular Crystals
with Heavy-atom Reagents

18 November 1998

1. Introduction.
   The description given below is adapted from a portion of the 1999 revision of the Industrial Macromolecular Crystallography Association Collaborative Access Team Environmental, Safety, and Health Plan. It is intended to provide a definition of the procedures to be followed by scientists and technicians at the IMCA-CAT facilities for the preparation of heavy-atom derivatives of their macromolecular crystals.
   
   
2. Purpose.
   When a macromolecular crystallographer wishes to determine a structure de novo where no homologous structure exists, he or she will employ one of two techniques: multiwavelength anomalous diffraction (MAD) and multiple isomorphous replacement (MIR). In the first of these techniques, atoms with absorption edges in the range of X-ray energies accessible to the users are incorporated into the macromolecule of interest by chemical or biochemical modification of the macromolecule prior to crystal growth or by exposing the already-grown crystals to a reagent containing the absorbing atom. Crystallographic data are then collected at several wavelengths (typically three or four) in the vicinity of the absorption edge. In the second technique, crystals are exposed (again, either before or after growth) to two or more heavy-atom reagents that produce substantial changes in the ordinary and the anomalous scattering of the sample. Thus many experiments employing MAD and MIR require a method for exposing an already-grown macromolecular crystal to a small quantity of a solution or a solid preparation of a heavy-atom reagent. The purpose of this Standard Operating Procedure is to define a safe and effective way to do this.
   
   
3. General safety remarks.
   Most, but not all, heavy-atom compounds are moderately toxic. The procedures outlined here minimize the likelihood of exposure to the compound for the scientist using it or passersby. They will also minimize problems associated with waste generated in exposing macromolecular samples to the heavy-atom reagents. The IMCA-CAT hood, located in the IMCA-CAT biochemistry laboratory, is an appropriate location for manipulation of heavy-atom reagents and for exposing macromolecular crystals to them. The IMCA-CAT facilities have not been certified for the manipulation of radioactive heavy-atom reagents or any other radioactive compounds, so these procedures exclude use of such compounds at this point. Therefore soaks with uranium or thorium compounds may not be performed at IMCA-CAT currently.

   Any heavy-atom reagent transported to or handled in the IMCA-CAT facility must have a Materials Safety Data Sheet (MSDS) on file in the IMCA-CAT MSDS binder, located just outside the west door of the biochemistry laboratory. The user should request that the IMCA-CAT administrative associate, Virginia Brown, obtain a material safety data sheet (MSDS) for each of the compounds that have been selected for use prior to bringing the chemical on site. The user should read and understand the MSDS sections on toxicity and treatment in case of an accident.

   Most heavy-atom compounds are water soluble, but for those that are not, a scientist must be particularly cautious since organic solutions can penetrate skin or create toxic vapors.

   All manipulation of heavy-atom reagents within the IMCA-CAT facilities, including the derivatization process itself, must be performed in the IMCA-CAT fume hood in the biochemistry laboratory on top of disposable laboratory paper. The only exception to this rule of "in the hood, on the diaper" is use of the laboratory balance to weigh out solid or liquid heavy-atom reagents. Particular care must be exercised in weighing out reagents, as described below. Lisa Keefe, the IMCA-CAT staff person in charge of the biochemistry laboratory, will provide the paper for the user and will arrange for disposal of the paper as hazardous waste after use.
   
   

4. Transport and storage.
   Store all heavy-atom compounds either in a chemical hood or a well-ventilated cabinet. Reagents to be stored at the IMCA-CAT facilities over lengthy periods (longer than a single visit by a research group) will be stored in a limited-access cabinet dedicated to the purpose and kept in the IMCA-CAT hood. Reagents brought in by users for a single visit to the IMCA-CAT facilities must be transported in a double-container and must be kept in the IMCA-CAT hood upon arrival. All transport of heavy-atom reagents must be conducted in accordance with Department of Energy transport regulations while on DOE property and with Department of Transportation regulations outside of DOE property.
   
   
5. 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; contact the Safety Coordinator to determine the appropriate procedure for dealing with a particular compound. When weighing out solids a safe procedure to follow is:
   1. Tare an appropriately sized container for the amount of solid/solution you will need
   2. With gloved hands, open the compound container in a functioning chemical hood and using a clean spatula carefully transfer enough of the solid to the tared vial to meet your requirements.
   3. Cap the bottle and the vial and wipe down the spatula with a Kimwipe
   4. Place the Kimwipe into the hazardous waste disposal container and the spatula in the wash bucket.
   5. Reweigh the vial to determine the actual amount of solid present and add water or solvent to dissolve the solid. Store the stock heavy-atom solution in the hood, either in the user-supplied double container or the CAT storage cabinet.
   6. Dispose gloves into hazardous waste.

   
   
   

6. Procedure for soaking heavy-atom reagents into macromolecular crystals.
   Soaking is a technique appropriate to manipulation of stable, preformed crystals that can be placed in artifical mother liquors containing various concentrations of heavy-atom compounds. To keep the volumes of heavy-atom reagent that will be added to the crystal mother liquor small (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 specially designated Hamilton syringe 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.
   
   
7. 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)

   Excluded from the table are uranyl and thorium reagents.

   Heavy-atom Reagent	Molecular Weight	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
   Ethyl mercurithiosalicylate (Thimerosal)		0.8mM	10-30 days	22
   (K/Na)AuCl4				22
   (Na/K)3IrCl6		5mM		21
   CH3CH2HgPO4				20
   K2PtCl6		5mM	-	19
   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

   
   
   

8. Exclusions.
   
   • Radioactive samples.
     At the present time, the IMCA-CAT laboratory facilities are unable to handle chemistry involving radioactive substances such as ³²P, ³⁵S, ¹²⁵I. Reactions involving compounds labelled with these radioactive nuclides should be performed in the home laboratories. As stated in the IMCA-CAT ES&H Plan, the level of radioactivity actually contained in a crystal prepared in this manner will in general be below the levels defined by the Department of Energy set forth in Chapter 5.2, Table 02-3 of the Argonne Environmental, Safety, and Health Manual. In these quantities the samples do not evince special handling requirements and therefore can be transported to and used at the IMCA-CAT facilities. However, the heavy-atom reagents themselves may not be brought into the IMCA-CAT facilities.
   • Volatile heavy-atoms. At the present time, the laboratory facilities under the supervision of IMCA-CAT is unable to guarantee the safe handling of extremely volatile heavy-atom compounds such as dimethyl mercury, tetramethyl lead, and tetraethyl lead. Storage and use of these compounds in the IMCA-CAT domain is forbidden until further notice. Final crystals for diffraction analysis, prepared in the home laboratories and containing containing any of these compounds will be permitted provided that the total concentration of toxic substance present falls below the maximum allowable levels recommended by APS E,S &H. If this is not the case, it will be necessary to review transport and use procedures to see that they can be safely handled and disposed of. Review of these quantities has not been completed, so until such review is completed, crystals containing these compounds should not be brought into or used at the IMCA-CAT facilities.
   • Pathogens. At no time will pathogenic organisms of any sort be allowed within the IMCA-CAT domain. IMCA-CAT have neither the facilities nor the expertise to handle these types of samples. The experimenter will be prevented from bringing samples of this type into the area supervised by IMCA-CAT.

   
   
   

9. References.
   Rould, M.A. "Screening for Heavy-Atom Derivatives and Obtaining Accurate Isomorphous Differences" Methods in Enzymology Vol 276 Part A: 461-472 (1997) or Blundell, T.L.; Johnson, L.N. "Preparation of Heavy Atom Derivatives" Protein Crystallography Chapter 8: 183-239 (1976) and references contained therein for a more thorough discussion of the preparation of heavy-atom derivative crystals.)

updated by Virginia Brown on 4 October 1999