Tables for
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 12.1, pp. 247-255   | 1 | 2 |
doi: 10.1107/97809553602060000679

Chapter 12.1. The preparation of heavy-atom derivatives of protein crystals for use in multiple isomorphous replacement and anomalous scattering

D. Carvin,a S. A. Islam,b M. J. E. Sternbergb and T. L. Blundellc*

aBiomolecular Modelling Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, England,bInstitute of Cancer Research, 44 Lincoln's Inn Fields, London WC2A 3PX, England, and cDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, England
Correspondence e-mail:

In this chapter an introduction to the heavy-atom data bank is provided and strategies are reviewed that can be adopted in the preparation of heavy-atom derivatives of protein crystals for use in multiple isomorphous replacement and anomalous scattering. Topics covered include: heavy-atom derivatives and their complexes; amino acids as ligands; the protein chemistry of heavy-atom reagents; metal-ion replacement in metalloproteins; analogues of amino acids; and the use of the heavy-atom data bank to select derivatives.

12.1.1. Introduction

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The traditional method of multiple isomorphous replacement (MIR) was introduced by Perutz and co-workers in 1954 (Green et al., 1954[link]) and is often enhanced by anomalous scattering (MIRAS) [see Blundell & Johnson (1976[link]) for a review]. The method remains popular for solution of the phase problem in the absence of the structure of a close homologue, although the use of multiple anomalous dispersion is likely to increase in the coming years (Hendrickson, 1985).

Protein crystals comprise an open lattice of protein molecules with solvent occupying the channels and spaces which normally comprise between 30 and 80% of the crystal volume. The preparation of a useful derivative requires the binding of a heavy atom to a specific position, usually on the protein surface, for example by the displacement of a lighter solvent molecule or an ion, without distorting the protein or crystal lattice.

Ideally, rational selection of suitable heavy-atom reagents requires a comprehensive knowledge and understanding of the crystalline structure of the protein. Normally, this information is unavailable since it is the objective of the crystal structure analysis! Nevertheless, the sequence and mechanism of action may suggest which heavy-atom reagents might be employed. There are reports in the literature of many attempts to make synthetic analogues of specific amino acids, by substituting selenium for sulfur residues in a chemically synthesized polypeptide or by removing an amino-terminal residue by the Edman technique and replacing it with an amino acid modified by a heavy atom [see Blundell & Johnson (1976[link]) for a review]. Alternatively, analogues of the substrate of an enzyme or carrier protein can sometimes be modified with a heavy atom; however, this will disturb the active site, which is usually the region of greatest interest to the structural biologist. Such methods have not proved very useful and will not be described further here. Most proteins studied now are recombinant; site-directed mutagenesis can replace methionines in the sequence, which occur on average once every fifty residues, by selenomethionines (Hendrickson et al., 1990[link]) or more recently by telluromethionines (Budisa et al., 1997[link]). Such approaches have revolutionized macromolecular crystallography through the use of anomalous-dispersion techniques, but have yet to provide a very efficient method of introducing atoms heavier than selenium into proteins.

Thus, the vast majority of successful heavy-atom derivatives employed in crystallographic analyses are obtained on a trial-and-error basis. In earlier studies, the protein was often covalently modified, purified and characterized before crystallization. There are some useful covalent modifications, for example, the reaction of mercury with the sulfhydryl groups of cysteinyl side chains and the iodination of tyrosyl side chains. The replacement of a metal-ion cofactor, such as calcium or zinc, can also give a useful derivative. However, pre-reaction of the protein often gives rise to conformational changes in the protein, and crystallization frequently occurs in a different or non-isomorphous form.

Most heavy-atom derivatives are produced by direct soaking of the crystals in a solution of the heavy-atom compound. With this approach, heavy-atom substitution patterns tend to be complex, with sites frequently only partially occupied. The specificity is often determined by entropic factors. Thus, sites between molecules in the crystal lattice, or between several different side chains brought together by the tertiary structure, may bind the metal ion, even if the side chains individually do not have strong affinity for the metal. Chelation is entropically driven, and bonds may form with unusual protein ligands, a major factor causing lack of specificity.

Blake (1968[link]) reviewed the data available for heavy-atom binding to proteins and suggested some generalizations. These were extended in a comprehensive review of protein heavy-atom derivatives (Blundell & Johnson, 1976[link]; Blundell & Jenkins, 1977[link]) which analysed the dependence of reactivity on protein side chain identity, nature of the reagent, pH, concentration, buffer etc. Over the past two decades, there have been discussions of the binding of some particular metal ions, but there have been no comprehensive analyses. Furthermore, protein–heavy-atom interactions have not often been fully described in publications of protein crystallographic analyses, and, in any case, the information has not been available in a format that could be used for systematic computer-based analysis.

We have now collected, either from the literature or directly from protein crystallographers, information on the preparation and characterization of heavy-atom derivatives of protein crystals. We have defined heavy atoms as those with an atomic weight greater than that of rubidium. We have assembled the information in the form of a data bank (Carvin et al., 1991[link]; Islam et al., 1998[link]) in which the coordinate data for the heavy-atom positions are compatible with the crystallographic data in the Protein Data Bank (Bernstein et al., 1977[link]). The data bank contains a wealth of information and provides the basis for further, more detailed analyses of heavy-atom binding to proteins. The information can be directly accessed and should be useful to protein crystallographers seeking to improve their success in preparing heavy-atom derivatives for isomorphous replacement and anomalous dispersion.

In this chapter we provide an introduction to the data bank and we review strategies that can be adopted in the preparation of heavy-atom derivatives of protein crystals for use in MIRAS.

12.1.2. Heavy-atom data bank

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The heavy-atom data bank (HAD) is a computer-based archival file system that contains experimental and derived information from successful multiple isomorphous replacement analyses in the determination of protein crystal structures. HAD is available at . The data bank makes available information which is otherwise only accessible in a widely distributed and fragmented form throughout the scientific literature or even unpublished in laboratory files.

The data bank contains information on heavy-atom derivatives for 969 protein crystals, 600 of which are deposited in the Protein Data Bank (PDB). A further 200 proteins are being processed at present. It contains information on the physical and chemical characteristics of each chemical compound that has proved successful in past protein crystallographic analyses: this includes the IUPAC name, trivial name, molecular formula, oxidation state, solution chemistry and stereochemistry. Experimental details of the preparation of the heavy-atom derivatives include the source of the protein, concentration of the heavy-atom solution, pH values, soak times and details of the buffer compositions used in the experiments; 3164 different experimental conditions are recorded. The atomic coordinates are given in the same format as the PDB coordinates for the 5500 binding sites of the heavy atoms. A statistical analysis is included for each of the 456 heavy-atom reagents; this includes range of pH values and a summary of the amino acids involved at the binding sites. For metalloproteins, it gives details of the type, number, geometry of coordination and function of the native metal(s) present. This is followed by a description of the procedure for native-metal substitution and details of the coordination of the substituted heavy atom. It also includes an extensive bibliography and references to other relevant web sites.

12.1.3. Properties of heavy-atom compounds and their complexes

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Potential ligands for heavy-atom reagents may be derived from the functional group(s) of reactive amino-acid side chains, from the buffer and from salting out/in agents. We must first consider factors that will influence the formation of such complexes in the environment of a protein crystal. Stability

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Ligands may be classified as either hard or soft. Hard ligands tend to be electronegative and interact electrostatically, with little delocalization of electron density. Water molecules, glutamates, aspartates, terminal carboxylates, and hydroxyl groups of serine and threonine from the protein, as well as acetate and citrate ions from the buffer, fall into this category. Conversely, soft ligands are polarizable and tend to form covalent bonds. Typical examples include the anions Cl, Br, I, S2−, CN, imidazole, methionine, cysteine, cystine and histidine from the protein. Ligands can be listed in series of increasing hardness: [ \def\emdash{-\!\!\!\!-} \eqalignno{ &\hbox{I}^{-} \gt \hbox{Br}^{-} \gt \hbox{Cl}^{-} \gg \hbox{F}^{-} \gg \hbox{H}_{2}\hbox{O},\cr &R\hbox{S}^{-} \gt R_{2}\hbox{S} \gg \hbox{NH}_{3} \gt \hbox{H}_{2}\hbox{O}, \cr &\hbox{CN}^{-} \gt R\hbox{NH}_{2} \gt \hbox{Cl}^{-} \gt \hbox{CO}_{2}^{-} \gt \emdash\!\hbox{OH of alcohol}. \cr}]

The metal components of the reagents may be classified as hard (class A) or soft (class B) in a similar way. Class A metals include the alkali metals, the alkaline earth metals, the lanthanide and actinide series, and the first-row transition metals from group III to group VA. Many of these metal ions have an inert-gas structure in which the electrons are held very strongly and tend to be non-polarizable. Metal ions in this class tend to interact with hard ligands, including the acetate, citrate and phosphate buffer components of mother liquor systems. On the other hand, class B metals have a preference for binding soft ligands. This group includes most members of the second and third row of the transition series (e.g. Ag, Cd, Pt, Au, Hg), which form cations such as [\hbox{Pt}(\hbox{NH}_{3})_{4}^{2+}] or anions such as [\hbox{Au}(\hbox{CN})_{2}^{-}], [\hbox{PtCl}_{4}^{2-}] and [\hbox{HgI}_{4}^{2-}]. The easily polarizable d electrons allow formation of covalent bonds with methionine, cysteine and imidazole, so displacing the ligands of the complexes. In the middle and towards the end of the first transition-metal series, the ions have properties intermediate between class A and B metals. Class B character increases in the series: [ \hbox{Fe}^{2+} \lt \hbox{Co}^{2+} \lt \hbox{Ni}^{2+} \lt \hbox{Cu}^{2+} \lt \hbox{Zn}^{2+}.] Thus, zinc binds to the polarizable sulfur of cysteine and imidazole of histidine as well as to carboxylates and water molecules. Tl+ and Pb2+, which each have an inert pair of electrons in their outer shell, are stable cations and prefer carboxylate rather than sulfur ligands or imidazole. Lability

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The rates at which ligands enter and leave a metal complex are important in the formation of heavy-atom derivatives, especially the covalent complexes of mercury, gold and platinum. The rate-determining step in unimolecular SN1 reactions is the expulsion of the leaving ligand from the metal complexes, which often proceeds relatively slowly. The intermediate complex, once formed, reacts with the entering ligand almost instantly. For SN1 reactions, the rate is directly proportional to the intermediate complex concentration but independent of the ligand concentration. The bimolecular SN2 mechanism involves attack by the ligand on the metal complex to form an intermediate complex, which then ejects the displaced ligand. The rate of reaction is proportional to the concentration of the initial species and the concentration of the nucleophile. SN2 reaction rates are dependent on the nature of the leaving group and the attacking nucleophile in the following ways: [ \eqalignno{ &\hbox{Relative rates of attack: } R\hbox{S}^{-} \gt \hbox{I}^{-} \gt \hbox{Br}^{-} \gt \hbox{NH}_{3} \gt \hbox{Cl}^{-} \gt R\hbox{O}^{-}\hbox{;} \cr &\hbox{Rate of leaving group: } \hbox{H}_{2}\hbox{O} \gt \hbox{Cl}^{-} \gt \hbox{NO}_{2}^{-} \gt \hbox{CN}^{-}.\cr}]

Sulfur ligands are good nucleophiles but poor leaving groups. They form thermodynamically stable complexes. The rate of leaving is influenced by the trans effect in square-planar complexes of Au(III) and Pt(II). Thus groups in square-planar complexes trans to NH3 are difficult to displace. This has implications for attempts to make derivatives of proteins in ammonium sulfate, where ligands may be replaced by NH3.

Rates of reaction depend not only upon which ligands are present in a heavy-atom complex but also on the character of the metal. For example, [\hbox{PtCl}_{4}^{2-}], [\hbox{AuCl}_{4}^{-}] and [\hbox{PdCl}_{4}^{-}] have similar square-planar geometries (Petsko et al., 1978[link]), but the rates of substitution vary: [ \hbox{PdCl}_{4}^{-} \gt \hbox{PtCl}_{4}^{2-} \gt \hbox{AuCl}_{4}^{-}.] Thus, if the reaction between the protein and a palladium or platinum complex is proceeding too fast, a gold derivative might be investigated. Oxidation state of metal ions in protein crystals

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In the environment of a living cell, the following oxidation states tend to be stable: [ \hbox{Os(II)}, \hbox{ Ru(II)}, \hbox{ Ir(III)}, \hbox{ Rh(III)}, \hbox{ Pt(II)}, \hbox{ Pd(II)}, \hbox{ Au(I)}.] Effect of pH

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Although the pKa of an individual amino acid in solution is generally defined within narrow limits, environmental and steric factors give rise to a wide range of values in proteins. Thus, the hydrogen-ion concentration influences the thermodynamic and kinetic stability of potential complexes. Protons compete with heavy-atom ions for the available binding site(s) on the protein. For example, below pH 3.5, cations bind less well to aspartic and glutamic acids due to the protonation of the carboxylate groups.

The nucleophilicity of histidine increases when it loses its proton, and thus its positive charge changes from around pH 6.0 to 7.0. Similarly, the nucleophilicity of cysteine increases dramatically when the thiolate ion is formed at pH ∼ 8.0. The thiolate ion is a stronger nucleophile than the thioether group of methionine, but when it becomes protonated it is considerably less effective. The nucleophilicity of the attacking groups varies in the order [ R\hbox{S}^{-} \gt R_{2}\hbox{S} \gt R\hbox{SH}.] Thus the number and occupancy of sites can be manipulated by varying the pH, often after cross-linking the crystals to stabilize them.

Extremes in pH can give rise to considerable difficulties in establishing suitable derivatives, as hydrogen and hydroxyl ions compete with the metal ion/complex for the protein and with the protein for the metal ion/complex. At extremely high pH values metals in solution tend to form insoluble hydroxides. The ranges of pH values that are useful for metal ions are given in Table[link].

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Useful pH ranges of some heavy-atom reagents derived from the heavy-atom data bank

No. of entriesMinimumAverageMaximumCompound
159 3.0 6.7 9.1 Potassium tetrachloroplatinum(II)
63 4.2 6.6 9.0 Potassium dicyanoaurate(I)
53 4.2 6.9 9.5 Mercury(II) chloride
59 2.8 6.7 9.0 Mercury(II) acetate
52 4.7 6.7 9.3 4-(Chloromercurio)benezenesulfonic acid
57 2.0 6.5 9.3 Potassium tetraiodomercurate(II)
36 5.4 6.7 8.5 Ethylmercurythiosalicylate (EMTS)
46 4.0 6.0 8.0 Potassium pentafluorooxyuranate(VI)
2 8.2 8.4 8.5 Barium(II) chloride
22 4.0 6.2 8.1 Lead(II) acetate
13 4.5 6.6 7.5 Lead(II) nitrate
1 6.5 6.5 6.5 Strontium(II) acetate
3 6.3 6.8 7.5 Thallium(I) acetate
2 5.9 6.6 7.2 Thallium(III) chloride
5 5.0 5.8 6.8 Gadolinium(III) chloride
9 4.9 6.7 7.5 Samarium(III) nitrate
7 4.9 6.6 8.7 Neodymium(III) chloride
64 4.1 6.3 8.6 Uranium(VI) oxyacetate

Varying the reactivity of amino-acid side chains by manipulation of the pH can enable the same heavy-atom ion/complex to bind at different sites, thus producing more than one derivative useful for phase determination. Effect of precipitants and buffers on heavy-atom binding

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Components present in the heavy-atom solution can have a profound effect on protein–heavy-atom interactions. The salting in/out agent (precipitant) and buffer are the principal sources of alternative ligands for the heavy-atom reagents, while protons compete with the heavy-atom ion/complex for the reactive amino-acid side chains.

Ammonium sulfate is the most successful precipitant in protein crystallization experiments (Gilliland et al., 1994[link]). However, its continued presence in the mother liquor can cause problems by interfering with protein–heavy-atom interactions. At high hydrogen-ion concentrations, the NH3 group is protonated (i.e. [\hbox{NH}_{4}^{+}]), but as the pH rises the proton is lost, typically around pH 6.0–7.0, enabling the group to compete with the protein for the heavy-atom reagent by an SN2 reaction.

The nucleophilic strength of potential ligands follows the order [ \hbox{NH}_{3} \gt \hbox{Cl}^{-} \gt \hbox{H}_{2}\hbox{O}.] The anionic complex [\hbox{PtCl}_{4}^{2-}] is present in excess ammonia at pH > 7.0 and it will react: [ \hbox{PtCl}_{4}^{2-} \rightarrow cis\hbox{-PtCl}_{2}(\hbox{NH}_{3})_{2} \rightarrow \hbox{Pt}(\hbox{NH}_{3})_{4}^{2+}.]

The resultant cationic complex is less susceptible to reaction due to the trans effect of NH3. Pd, Au, Ag and Hg complexes react in a similar way. Decreasing the pH of the solution reduces the amount of free ammonia available through protonation (Sigler & Blow, 1965[link]). Such a technique may give rise to other problems (e.g. cracked crystal, decreased nucleophilicity of the protein ligands).

Changing the precipitant to sodium/potassium phosphate or magnesium sulfate may alleviate the situation, but it may also present other problems. For instance, [\hbox{PO}_{4}^{3-}] displaces Cl from [\hbox{PtCl}_{4}^{2-}], thus increasing the negative charge. Both [\hbox{PO}_{4}^{3-}] and [\hbox{SO}_{4}^{2-}] form insoluble complexes with class A metals (e.g. lanthanide and uranyl cations) (Petsko et al., 1978[link]). Both acetate and citrate form complexes with class A metals, but citrate, a chelating ion, binds more strongly. Tris buffer is probably preferable; it binds many cations, but the complexes formed tend to be relatively unstable. Solubility of heavy-atom compounds

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The solubility of a heavy-atom compound will depend upon the precipitant, buffer and pH. Typically, the component present in the highest concentration is the precipitant, either as salts (e.g. ammonium sulfate) or as an organic-based reagent (e.g. ethanol, MPD, PEG). Heavy-atom compounds that are essentially covalent and organic in character will be more soluble in ethanol, MPD, PEGs and other organic precipitants.

Although the solubility of tetrakis(acetoxymercurio)methane (TAMM) is higher than most multiple-heavy-atom compounds in aqueous solutions, the presence of glycylglycine or charged mercaptans, such as cysteamine or penicillamine, can increase solubility further (Lipka et al., 1976[link]). The ratio of TAMM to solubilization agent (e.g. glycylglycine) is typically 1:10. Even so, the final solubility of TAMM depends on the concentration of competing anions (e.g. chloride) (O'Halloran et al., 1987[link]).

Many organometallic compounds are relatively insoluble in aqueous solutions, but their solubility may be increased by pre-dissolving in an aprotic solvent such as acetonitrile.

Iodine and several inorganic iodide salts are insoluble in aqueous solutions. This can be rectified by dissolving the heavy-atom compounds in an aqueous solution of KI. Effect of concentration, time of soak and temperature on heavy-atom binding

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Most heavy-atom derivatives are prepared by diffusing or dialysing the compound into the crystals. Concentrations have typically ranged from 0.1–100.0 mM. Occasionally, concentrations as low as 0.001 mM have been employed to maintain crystal integrity. Low concentrations favour sites where the interactions between the heavy atom and the protein ligands are strongest. Decreasing the number of non-specific interactions minimizes the amount of heavy-atom reagent in the lattice. The latter absorbs X-rays without contributing to the diffraction pattern except at low angles. Increasing the concentration may give rise to other binding site(s). Usually, the higher the concentration employed, the shorter the soak time required for equivalent substitution. Short soak times at high concentrations tend to denature the crystals more often than long soaks at low concentrations. At very high concentrations (i.e. > 100 mM), the heavy-atom compound perturbs the protein crystal–mother liquor equilibrium by withdrawing water molecules from the hydration shell around the periphery of the crystal. Disorder of the crystals can sometimes be avoided by the application of a cross-linking reagent (e.g. glutaraldehyde). The optimal concentration is the lowest concentration that consistently reproduces intensity differences in the diffraction pattern of 15–25% without cracking and disordering the crystals.

Length of soak may be important. The heavy-atom data bank shows that, typically, soak times range from one day to one week. Useful derivatives have been prepared with a soak time of an hour to over a year. If no binding is apparent after several days, extending the soak time to over a week may produce some binding, but this is rare. Soaks of 24 hours for simple inorganic salts and up to one week for other types of heavy-atom compounds will normally suffice when screening for binding. The concentration of the heavy-metal compound that can be achieved will depend on its solubility in the crystal stabilization solution. Normally, the longer the soak, the greater the occupancy. Exceptions can arise due to undesirable chemical reactions between components present in the derivatization solution.

For covalent-bond formation, the length of soak and the concentration can be short (e.g. 1 h, 0.01 mM). This is especially true for mercury derivatives of proteins that have reactive sulfhydryls (Ringe et al., 1983[link]).

Variations in the temperature can also alter the rate of reaction. The UO2 acetate derivative of rhombohedral insulin binds twenty times more slowly at 4 °C than at ambient temperature (Blundell, 1968[link]). A lower temperature allows greater control over the rate of substitution. Conversely, heavy-atom derivatives that do not appear to bind may do so upon elevation of the temperature.

12.1.4. Amino acids as ligands

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The reactivity of the heavy-atom reagent will also depend on the state of the amino-acid residues in the protein.

The thiolate anion of cysteine, a potent nucleophile, reacts almost irreversibly with mercuric complexes or organomercurials. It also acts as a fast-entering attacking group in SN2 ligand substitution reactions with other class B metals (e.g. Ag, Ir, Rh, Pt, Pd, Au), forming stable complexes. Below pH 6, the thiolate anion becomes protonated. As covalent reactions are less sensitive to hydrogen-ion concentration than ligand substitution reactions, cysteines still bind rapidly with mercurials, but there is negligible reaction with other class B metals (Petsko et al., 1978[link]).

Cystines are very weakly reactive in ligand substitution reactions. However, [\hbox{PtCl}_{4}^{2-}] binds to disulfides in some proteins with displacement of a chloride ion (Lipscomb et al., 1970[link]; Sigler et al., 1968[link]). Mercurials rarely insert spontaneously into disulfide linkages. However, substitution of mercury can be achieved either by the prior application of a reducing agent such as dithiothreitol (Ely et al., 1973[link]; Sperling et al., 1969[link]), or by direct application of reducing mercurous ions (Sperling & Steinberg, 1974[link]).

The non-ionizable thioether group of methionine is unreactive towards mercurials, but the lone pair of electrons on sulfur allows nucleophilic SN2 ligand substitution. Methionine will displace Cl, I, Br and NO2 ligands from platinum complexes to form a stable bond. The reaction of methionine with platinum compounds is not pH sensitive within the normal range. The residue may become unreactive through oxidation, first to the sulfoxide and then to the sulfone; only the sulfoxide can be reduced readily by thiols or other reducing agents.

Below pH 6, histidine exists mainly as an imidazolium cation. Although this is not reactive as a nucleophile, it can interact electrostatically with anionic complexes. At pH 7 and above, the unprotonated imidazole is a good nucleophile, being able to displace Cl, Br, I and NO2 ligands from platinum, silver, mercury and gold complexes. Electrophilic substitution of iodine in the imidazole ring is feasible, but the conditions are severe and it has not proved very useful in preparing derivatives.

At pH < 8.5, the [epsilon]-amino group of lysine is protonated, allowing it to form weak electrostatic interactions with anionic heavy-atom complexes, but not to participate in SN2 substitution reactions. Above pH 9, the free amino group can displace Cl but not Br, I or NO2 ligands from platinum and gold complexes. The pKa of the guanidinium group of arginine is very high (> 12 in proteins), so it interacts electrostatically as a cation with heavy-atom anionic complexes.

The indole ring of tryptophan is relatively inert to electrophilic substitution by iodine, but the ring nitrogen can be mercurated (Tsernoglou & Petsko, 1976[link]). The reaction is not pH dependent, but there should be no competing nucleophiles in the mother liquor. Tryptophan does not usually participate as a ligand in substitution of heavy-atom complexes.

The phenolate oxygen anion of tyrosine is a good nucleophile and has the potential to bind a substantial number of heavy-atom complexes via SN2 ligand substitution reactions. However, it has a very high pKa value of 10.5. Below pH 10, the protonated oxygen predominates, making electrophilic aromatic substitution by iodine the principal reaction.

Aspartic and glutamic acids have side-chain pKa values in the range 3 to 4. At low pH, they will be protonated and unreactive. Above pH 5, the side chains will be anionic, making them good ligands for class A cations such as uranyl and rare earths. Glutamine and asparagine take part in metal coordination but rarely bind strongly enough to metal ligands on their own.

Hydroxyl groups of serines and threonines are fully protonated at normal pH values and are consequently not reactive nucleophiles. Abnormally reactive serines, usually at the active site as in serine proteases and β-lactamases, can react with heavy-atom reagents to give useful derivatives.

12.1.5. Protein chemistry of heavy-atom reagents

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The heavy-atom data bank (Islam et al., 1998[link]) can be used to analyse the most commonly used heavy-atom reagents: these are given in Table[link]. This shows that platinum, gold, mercury and uranyl have provided the most useful reagents.

Table| top | pdf |
The 23 most commonly used heavy-atom reagents

The first column gives the number of times the reagent has been used in the analyses included in the heavy-atom data bank.

287 Potassium tetrachloroplatinum(II)
111 Potassium dicyanoaurate(I)
103 Uranyl acetate
101 Mercury(II) acetate
98 Mercury(II) chloride
85 Ethylmercurythiosalicylate (EMTS)
82 Potassium tetraiodomercurate(II)
81 para-Chloromercuriobenzenesulfonate (PCMBS)
75 Trimethyllead(IV) acetate
73 Potassium pentafluorooxyuranate(VI)
73 Phosphatotris(ethylmercury)
61 Potassium tetranitritoplatinum(II)
60 Uranyl nitrate
58 Potassium tetracyanoplatinum(II)
57 Dichlorodiammineplatinum(II)
51 Potassium hexachloroplatinum(IV)
51 Methylmercury chloride
44 Potassium tetrachloroaurate(III)
42 para-Chloromercurybenzoate (PCMB)
39 Lead(II) acetate

The heavy-atom data bank can be used as a source of information about the reactivity of proteins to different heavy-atom reagents. This provides the basis for the following analysis. Hard cations

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Uranyl-ion complexes have proved the most popular A-group metal reagents for preparing heavy-atom derivatives of protein crystals (see Table[link]). [\hbox{UO}_{2}^{2+}] is a linear, covalent group based on U(VI), the most stable oxidation state of uranium. Table[link] lists the most commonly used uranyl derivatives. Uranyl compounds may show 2 + 4, 2 + 5, or 2 + 6 coordination, with ligands lying in or near a plane normal to the O=U=O2+ axis. These equatorial ligands may be neutral (e.g. H2O) or anionic (e.g. [\hbox{NO}_{3}^{-}], CH3COO,oxalate2−, F, Cl1 or [\hbox{O}_{2}^{-}]); the nitrate and acetate are bidentate ligands. An example is given in Fig.[link]. Anionic complexes, such as [\hbox{UO}_{2}\hbox{F}_{5}^{3-}], have been found near negatively charged amino-acid residues (e.g. Glu and Asp), suggesting that the equatorial ligands have been displaced. At low pH, uranyl groups have been located near the hydroxyl groups of threonine and serine residues.

Table| top | pdf |
The five most popular uranium derivatives

The first column gives the number of times the reagent has been used in the analyses included in the heavy-atom data bank.

103 Uranyl acetate
73 Potassium pentafluorodioxyuranate(VI)
60 Uranyl nitrate
8 Uranium(VI) oxysulfate
4 Sodium triacetatedioxyuranate(VI)

Figure | top | pdf |

The binding site for uranyl ions in cytochrome b5 (oxidized: 3B5C). The positions of the ligands in the parent crystals are shown; these probably move in the complex.

The fifteen lanthanides have similar chemical properties and are generally used as nitrates, acetates or chlorides (Blundell & Johnson, 1976[link]; Carvin, 1986[link]). The lanthanide contraction, a steady decrease in size with increasing atomic number, allows selection of an ion with a radius that will give high occupancy and isomorphism. Gadolinium and samarium salts have the added advantage that the number of anomalous electrons is high.

Lanthanide ions have greater selectivity than the uranyl ion, which often forms clusters on the protein surface. Uranyl complexes and lanthanide ions are not very soluble above pH 7 and pH 9, respectively, due to the formation of hydroxides. Phosphate buffers should be avoided since they will compete for the heavy atom, often giving insoluble phosphates. In the presence of citrate, samarium is chelated and, since the citrate is difficult to replace, reaction may be inhibited. However, exchanging the buffer for Tris or acetate may enable a useful derivative to be obtained. Thallium and lead ions

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Thallium and lead can provide useful derivatives, especially in their lower oxidation states, Tl(I) and Pb(II), when they resemble class A metals. Owing to the non-group valence and presence of an inert pair of electrons, the ionic radii of Tl+ (1.44 Å) and Pb2+ (1.21 Å) are greater than most class A metals. Thallous and plumbous cations prefer carboxylate rather than imidazole or sulfur ligands, although Pb2+ occasionally manifests its intermediate character by interacting with imidazole groups. Thallic (Tl3+) and plumbic (Pb4+) ions are similar to class B metals, showing preferential binding to soft ligands, but they are easily reduced in protein solutions. B-metal reagents

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The most useful members of the B-metal group, platinum, gold and mercury, give rise to an extensive range of heavy-atom compounds which form covalent, electrostatic and van der Waals complexes with proteins. Some compounds can bind to the protein molecule in different ways; for example, [\hbox{PtCl}_{4}^{2-}] can bind either covalently to the thioether group of methionine or electrostatically with positively charged residues.

Mercury compounds have proved very successful for preparing heavy-atom derivatives of protein crystals (Table[link]), mainly due to the ease of formation of covalent bonds with cysteine residues. An example is given in Fig.[link] in which mercuric chloride has been used to replace zinc in thermolysin. Hg2+ complexes are commonly two-coordinate linear and four-coordinate tetrahedral. The most popular mercury reagents are given in Table[link]. The covalent character in Hg—L bonds, especially in the two-coordinate complexes, can cause solubility problems in aqueous solutions. However, an excess of an alkali metal salt (e.g. [\hbox{HgI}_{2} + 2\hbox{KI} \rightarrow \hbox{K}_{2}\hbox{HgI}_{4}]) will often convert the compound to a more soluble anionic complex of the type [\hbox{Hg}X_{4}^{2-}], where X = Cl, Br, I, SCN, NCS, CN, [\hbox{SO}_{4}^{2-}], oxalate2−, [\hbox{NO}_{3}^{-}] or [\hbox{NO}_{2}^{-}]. In the presence of ammonium salts at high pH values, the cationic tetraammine complex, [\hbox{Hg(NH}_{3})_{4}^{2+}], tends to form. Variation in the charge on the aromatic groups of organomercurials can give rise to different substitution patterns.

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The five most popular mercury derivatives

The first column gives the number of times the reagent has been used in the analyses included in the heavy-atom data bank.

101 Mercury(II) acetate
98 Mercury(II) chloride
85 Ethylmercurythiosalicylate (EMTS)
82 Potassium tetraiodomercurate(II)
81 para-Chloromercuriobenzenesulfonate (PCMBS)

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Mercuric ions replace zinc in thermolysin (3TLN). The mercuric ion is shown superposed on the parent crystal structure; notice that the mercuric ion is slightly displaced from the zinc position due to its larger ionic radius.

Silver, used as the nitrate, tends to interact with cysteine or histidine (see Fig.[link]). In the presence of ammonium sulfate, it probably reacts as the ammonia complex, [\hbox{Ag(NH}_{3})_{4}^{+}]. Silver ions are less polarizing and less reactive than Hg2+ ions; thus they give similar derivatives but often with less disorder, as in glucagon (Sasaki et al., 1975[link]). Where the metal ion displaces a proton, Ag+ will need to react at a higher pH than Hg2+.


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The binding of a silver ion to immunoglobulin Fab (2FB4). The positions of the ligands in the parent crystals are shown, and these must move in the complex to coordinate the silver ion.

The class B metals palladium, platinum and gold form stable covalent complexes with soft ligands, such as chloride, bromide, iodide, ammonia, imidazole and sulfur groups. The stereochemistry of their complexes depends on the number of d electrons present. For instance, the d10 ion of Au(I) gives a linear coordination of two [e.g. [\hbox{Au(CN)}_{2}^{-}]], whereas d8 ions of Pd(II), Pt(II) and Au(III) are predominantly square planar, giving cationic [e.g. [\hbox{Pt(NH}_{3})_{4}^{2+}]], anionic [e.g. [\hbox{Au(CN)}_{4}^{-}], [\hbox{PtCl}_{4}^{2-}] and [\hbox{PdCl}_{4}^{2-}]] or neutral [e.g. Pt(NH3)2Cl2] complexes. These may accept an additional ligand to give square pyramidal coordination or two ligands to give octahedral coordination. The additional ligands are normally more weakly bound. Pt(IV) has a d6 configuration and forms stable octahedral complexes, such as [\hbox{PtCl}_{6}^{2-}], with six equivalent covalently bound ligands.

The kinetic and thermodynamic stability of these complexes depends on the protein ligands, buffer, pH and salting in/out agent (Petsko et al., 1978[link]). Anionic groups do not readily react with anionic reagents, such as RS, but are attacked more readily by neutral nucleophiles such as RSH, R-imidazole or RNH2. The inert cationic group [\hbox{Pt(NH}_{3})_{4}^{2+}] is most likely to form electrostatic complexes with anionic groups, such as carboxylate. The neutral Pt(NH3)2Cl2 molecule, however, can penetrate into hydrophobic areas but requires a stronger nucleophile such as RS. In acidic and neutral solutions, [\hbox{PtCl}_{4}^{2-}] reacts most commonly with methionine (Figs.[link] and[link]), cystine (disulfide) (Fig.[link]), N-termini and histidine to form stable complexes. However, methionine reacts faster than histidine. Thus, it is possible to use time as a variable to define specificity. The most popular platinum reagents are listed in Table[link].

Table| top | pdf |
The five most popular platinum derivatives

The first column gives the number of times the reagent has been used the analyses included in the heavy-atom data bank.

287 Potassium tetrachloroplatinum(II)
61 Potassium tetranitritoplatinum(II)
58 Potassium tetracyanoplatinum(II)
57 Dichlorodiammineplatinum(II)
51 Potassium hexachloroplatinum(IV)

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The binding of [\hbox{PtCl}_{4}^{2-}] through a methionine in azurin (1AZU).


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The relative positions of methionine side chains (carbon: green; sulfur: yellow) in the parent crystals to the binding of platinum (pink) of [\hbox{PtCl}_{4}^{2-}]. The methionine side chains have been least-squares fitted.


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The relative positions of cystine disulfide bridges (carbon: green; sulfur: yellow) in the parent crystals to the binding of platinum (pink) of [\hbox{PtCl}_{4}^{2-}]. The cystine side chains have been least-squares fitted, and only those with torsion angles in the range [99.7 \pm 8.3^{\circ}] have been used.

In aqueous solution, the square-planar complex [\hbox{AuCl}_{4}^{-}] is hydrolysed to [\hbox{Au(OH)}_{4}^{-}] in about one hour, or in the presence of a protein, reduced to Au(I) by methionine. In ammonium sulfate it probably exists as AuCl3(NH3), [\hbox{AuCl}_{2}(\hbox{NH}_{3})_{2}^{+}] and [\hbox{Au(NH}_{3})_{4}^{3+}]. In contrast, [\hbox{Au(CN)}_{2}^{-}] is more stable and normally binds electrostatically. However, on occasions at pH > 6.0, the [\hbox{Au(CN)}_{2}^{-}] complex has bound to cysteine residues by nucleophilic displacement reactions.

Osmium resembles platinum in many ways and typically acts as a class B metal. It occurs in all oxidation states from 0 to VIII, but most usually in III, as in K3OsCl6; in IV, as in K2OsCl6; in VI, as in K2OsO2(OH)4; and in VIII, as in osmium tetraoxide, OsO4. Higher-oxidation-state compounds tend to be reduced to OsO2(OH)2 in most crystallization solutions and in the presence of ammonia or halide ion they can become further reduced to cationic or anionic complexes, such as [\hbox{Os}(\hbox{NH}_{3})_{6}^{3+}] or [\hbox{Os}\hbox{Cl}_{6}^{2-}] . Anionic complexes may be substituted by histidine residues at pH > 7.0 or bound as ion pairs by histidine at pH < 7.0 or protonated amino groups. Cationic complexes tend to bind to negatively charged residues via electrostatic interactions.

Iridium is found in all oxidation states from II to VI but commonly exists in III, as in K3IrCl6, and IV, as in (NH4)2IrCl6. Ir(III) is similar to rhodium(III) and is found in a variety of cationic, uncharged and anionic complexes. All Ir(III) complexes are kinetically inert, whereas most anionic complexes of Rh(III) are labile. Ir(IV) is commonly found as the hexahalo complexes [\hbox{Ir}X_{6}^{2-}] (except iodine), which are also fairly kinetically inert. Cationic [e.g. [\rm{Ir(NH}_{3})_{6}^{3+}]], neutral (i.e. IrCl3) and anionic (i.e. [\hbox{IrCl}_{6}^{2-}]) species have proved useful in forming derivatives of protein crystals. Electrostatic binding of heavy-atom anions

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Positively charged groups of proteins, such as the α-amino terminus, [epsilon]-amino of lysine, guanidinium of arginine and imadazolium of histidine, may form ion pairs with heavy-atom anionic complexes. For example, [\hbox{HgI}_{4}^{2-}] and [\hbox{HgI}_{3}^{-}] can bind through electrostatic interactions. Anionic metal cyanide complexes tend to be more resistant to substitution and consequently interact electrostatically on most occasions. For example, [\hbox{Pt(CN)}_{4}^{2-}] binds at several sites involving lysine or arginine residues in proteins (Fig.[link]). [\hbox{Pt(CN)}_{4}^{2-}] and [\hbox{Au(CN)}_{2}^{-}] can act as inhibitors by binding at coenzyme phosphate sites.


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The binding of [\hbox{Pt(CN)}_{4}^{2-}] to aldose dehydrogenase (8ADH). Hydrophobic heavy-atom reagents

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Since many heavy-atom reagents are hydrophilic, most interactions occur at the protein surface. However, substitution, addition or removal of the non-heavy-atom component(s) of the reagent can alter the hydrophilic–hydrophobic balance and lead to penetration of the core. For example, anionic complexes such as [\hbox{HgCl}_{4}^{2-}] and [\hbox{PbCl}_{6}^{2-}] are hydrophilic and would not normally enter the protein core, although organometallics, such as RHgCl and R3PbCl (R = aliphatic or aromatic), are much more hydrophobic and can do so.

Hydrophobic organomercury compounds of the general formula RHgX, where R is an aliphatic or aromatic organic group, react with sulfhydryls through displacement of X. When X is [\hbox{PO}_{4}^{3-}], [\hbox{ SO}_{4}^{2-}] or [\hbox{NO}_{3}^{-}], the bond is ionic, making the formation of the cation RHg+ easier. R is often chosen to be a small aliphatic group (e.g. CH3, C2H5). However, the presence of a benzene ring enhances the stability of the heavy-atom reagent. Careful selection of the X group can assist penetration into the hydrophobic core. The hydrophobicity of X follows the order [ \hbox{PO}_{4}^{3-} \lt \hbox{NO}_{3}^{-} \lt \hbox{Cl} \lt \hbox{Br} \lt \hbox{I} \lt R.] RHgR (R = aliphatic or aromatic) compounds also bind sulfhydryl residues in hydrophobic regions. The mechanism of reaction of methylphenylmercury with buried sulfhydryl groups may involve fast dissolution in the hydrophobic interior of the protein followed by a slow reaction with neighbouring sulfhydryl residues (Abraham et al., 1983[link]). They are difficult to prepare in aqueous solutions; an aprotic solvent, such as acetonitrile, can improve solubility, but this is not normally a problem in high concentrations of organic components, such as PEG, MPD or ethanol.

Inert gases were first used in the analysis of myoglobin. Schoenborn et al. (1965[link]) discovered that the hydrophobic site that bound [\hbox{HgI}_{3}^{-}] also bound a xenon atom at 2.5 atmospheres. They proposed that this may be a general way of producing heavy-atom derivatives of proteins. Recently, there has been increasing interest in this idea, which has now been developed to produce well defined derivatives of a wide range of different proteins. Crystals are subjected to high gas pressures. Xenon requires about 10 atmospheres in order to get saturated binding sites. Krypton binds much less strongly and requires around 60 atmospheres. Since the binding of both inert gases is reversible, it is necessary to keep the protein crystals in a gaseous environment in a specialized pressure cell. Such pressure cells have been developed by Schiltz (1997[link]) at LURE. Xenon binds to hydrophobic cavities, with little conformational change and a retention of isomorphism in crystals. Krypton binds at the same sites as xenon, but since it is lighter and needs higher pressure it has been exploited less by protein crystallographers. However, it has a well defined K edge at around 1 Å and so has attractions for multiple-wavelength anomalous dispersion. Iodine

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In addition to their use in isomorphous replacement, iodine derivatives of crystalline proteins have been prepared as tyrosine or histidine markers to assist main-chain tracing and to act as a probe for surface residues. The order of reactivity towards these reactive residues is [ \hbox{Tyr} \gt \hbox{His} \gg \hbox{Trp}.] [\hbox{I}_{3}^{-}], I, I+ and I2 can be generated by several different methods. An equimolar solution of KI/I2 or NaI/I2 in 5% (v/v) ethanol/water solution is often used to generate the anionic species [\hbox{I}_{3}^{-}] and I. An oxidizing agent, such as chloramine T, can be added to KI, typically in a concentration ratio of 1:50; alternatively, polystyrene beads derivatized with N-chlorobenzene sulfonamide can be used with NaI. Similarly, the addition of excess KI to ICl or OI will generate [\hbox{I}_{3}^{-}], I and I+. To avoid oxidation of iodine solutions, the pH should be less than 5.0. To avoid cracking the crystals, it may be necessary to increase the iodine concentration very slowly and to wash the derivatized crystals in the mother liquor in order to remove free I2. Mono- or di-iodination of tyrosines can cause disruption of the protein structure either because of the larger size or the breaking of hydrogen bonds due to lowering of the pKa of the phenolic hydroxyl. Polynuclear reagents

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The structure determination of large multicomponent systems such as the 50S ribosomal subunit (Yonath et al., 1986[link]) or the nucleosome core particle (O'Halloran et al., 1987[link]) requires the addition of reagents with a greater number of electrons, preferably in a compact polynuclear structure. Such reagents may be either cluster compounds or multimetal centres having metal–metal bonds.

Polynuclear reagents should preferably be covalently bound to one or a few specific sites, either first in solution or later in the crystals. Spacers of differing length can be inserted into the reagent to increase accessibility. Their low solubility in aqueous solutions can often be overcome by dissolving them in an apolar solvent (e.g. acetonitrile). Tetrakis(acetoxymercurio)methane (TAMM) and di-m-iodobis(ethylenediamine)diplatinum(II) nitrate (PIP) have better solubility in aqueous solutions than other polynuclear heavy-atom compounds.

Polynuclear heavy-atom reagents give an enhanced signal-to-noise ratio in low-resolution MIR studies, but this advantage is offset by the fall-off in scattering amplitude that arises from interference of diffracted waves at higher resolution. In the nucleosome core particle, the scattering reached 50% of its zero-angle value at 7.0 Å, while the relative drop for a single heavy atom was 10% (O'Halloran et al., 1987[link]). Cluster and multimetal reagents that have been successfully employed in protein structure determinations have been reviewed by Thygesen et al. (1996[link]).

12.1.6. Metal-ion replacement in metalloproteins

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The metal-ion cofactor can sometimes be displaced by dialysis or diffusion by a heavy-atom solution, but usually the cofactor is first removed by a chelating agent (e.g. EDTA) or by acidification. These are best carried out on the crystals. Alternatively, the metal can be substituted by biosynthesis of the metalloprotein under enriched conditions of the substituting metal, an approach which has been successful in displacing zinc with cobalt and other lighter metals.

The metal ions are best substituted by a metal of similar character and radius. Thus, calcium is an A-group metal which prefers ligands containing oxygen atoms that may originate from carboxylic, carboxyamide, hydroxyl, main-chain carbonyl groups and water molecules. Divalent alkaline earth metal ions (e.g. Sr2+, Ba2+) or trivalent lanthanide ions can bind at calcium sites but can give very different coordination geometry and stability. Nd3+ and Sm3+ can displace some Ca2+ ions with negligible change in structure (Fig.[link]). On the other hand, zinc has a relatively small ionic radius and is more polarizing. Structural zinc atoms are often tetrahedrally coordinated by cysteine residues, while those at active sites frequently bind histidine, often in association with a water molecule and/or carboxylate ligands. Cadmium or mercury can replace zinc, but often with a conformational change leading to lack of isomorphism.


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The displacement of calcium by samarium in thermolysin. The samarium of the heavy-atom derivative is shown superposed on the parent crystal structure.

12.1.7. Analogues of amino acids

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Attempts to replace amino acids by heavy-atom substituted synthetic analogues with a similar charge and shape have not proved successful in large proteins, although a selenocystine was used successfully in the analysis of oxytocin (Wood et al., 1986[link]). However, the production of proteins labelled by selenium using biological substitution of selenomethionine (SeMet) for methionine (Hendrickson, 1985[link]) has been stimulated by multiple-wavelength anomalous dispersion (MAD) (Hendrickson et al., 1990[link]). Methionine biosynthesis is blocked in the cells in which the protein is produced and SeMet is substituted for Met in the growth medium. The generality of the labelling scheme for proteins is the root of its success, as discussed by Doublié (1997[link]).

SeMet has been incorporated into proteins expressed in Escherichia coli strains that are auxotrophic for Met [strain DL421 (Hendrickson et al., 1990[link]); strain B834 (Leahy et al., 1994[link]); strain LE392 (Ceska et al., 1996[link])]. Nearly complete incorporation has also been reported in non-auxotrophic bacterial strains, E. coli strain XA90 (Labahn et al., 1996[link]), in a mammalian cell line (Lustbader et al., 1995[link]) and in baculovirus-infected insect cells (Chen & Bahl, 1991[link]). Usually, somewhat higher than normal concentrations of disulfide reducing agents, such as dithiothreitol or mercaptoethanol, are sufficient to protect SeMet from air oxidation to the selenoxide, although crystallization in an inert atmosphere may be necessary. Proteins usually have SeMet substituted for Met at levels approaching 100%. The cells are viable and the proteins are functional.

Site-directed mutagenesis offers an alternative approach for the introduction of specific heavy-atom binding sites. A common procedure is to replace residue(s) in the variable part of the primary structure with cysteine. The selection of the residue to mutate in a protein of unknown structure remains a challenge.

Although selenocysteine is toxic to cells, cysteine auxotrophic strains, in which proteins can be synthesized with the seleno derivative, have been developed (Miller, 1972[link]; Muller et al., 1994[link]). The bacteria are grown under limiting amounts of cysteine with no other sulfur source. They are induced for 10 min and then resuspended in selenocysteine for a 3 h incubation. The protein is purified with a reducing agent. In general, the substitution at the selenocysteine seems to be less satisfactory than selenomethionine, with occupancy often as low as 20%.

Budisa et al. (1997[link]) have experimented on incorporating a range of novel amino-acid analogues using in vitro suppression. This is achieved by suppressing the stop colons and engineering tRNA synthases to incorporate the analogue. Possible candidates are telluromethionine, 5-bromotryptophan, 5-iodotryptophan, selenotryptophan and tellurotryptophan. The bioincorporation of TeMet into derivatized crystals did not greatly affect their stability in buffer solutions and to X-radiation. Isomorphism was maintained despite the C—Te bond being longer than C—Se or C—S. TeMet crystals are not as suitable for MAD analysis as SeMet crystals due to the 0.3 Å absorption edge of tellurium. The method is restricted to methionine residues located in the hydrophobic regions, since solvent accessibility may cause undefined chemical reactions with the highly reactive C—Te side chain. Thus the protein must be expressed in the folded form.

12.1.8. Use of the heavy-atom data bank to select derivatives

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The heavy-atom data bank is probably best exploited by first investigating the most commonly used heavy-atom reagents with a view to obtaining mercury, platinum and uranyl derivatives that tend to bind at different sites. The most common reagents (Table[link]) can first be selected and tested for suitability in terms of amino-acid sequence, pH, buffer and salt. If there are many sulfhydryls, several mercurials might be exploited, or if there are several methionines, other platinum agents might be investigated. A high pH would argue against use of uranyl due to the insolubility of hydroxides; the presence of ammonium sulfate would argue for as low a pH as possible. The presence of citrate would imply changing the buffer for acetate if A-group metals, such as uranium or lanthanides, were to be used.

For each heavy-atom agent, the conditions of its previous use can be checked against the conditions of crystallization in the current study. Conversely, the database can be interrogated for reagents that have been used in similar conditions. In each case, derivatives that maximize the variety of ligands should be exploited.

The time of soak should be first set according to previous experience indicated in the database. However, the progress of heavy-atom substitution needs to be monitored by checking for change of colour, transparency or cracking. If cracking and disruption of the crystals occurs quickly, a less reactive reagent can be tried, and, conversely if substitution is insufficient, a more reactive reagent can be tried. If there are several cysteines, different derivatives can be obtained with mercurials of different size and hydrophobicity. In each circumstance, the data bank should provide useful information to assist in choosing reagents.

Please keep information about the heavy-atom binding sites and the heavy-atom structure-factor amplitudes. They should be submitted to the Protein Data Bank.


We thank all those who have generously sought out and sent us details of the heavy-atom binding sites in their derivatives, and the ICRF and the Wellcome Trust for support.


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