Page 101 - Macromolecular Crystallography

90 MACROMOLECULAR CRYS TALLOGRAPHY

6.3 Selection of heavy-atom reagents with proteins in a similar fashion, except for those
containing (Pt(CN) 2 ) 2− which bind to positively
Both the size and chemical composition of the
charged residues. Mercurial compounds are the sec-
molecule under investigation are important criteria
ond most successful group of reagents in derivative
to consider when selecting heavy-atom reagents for
preparation; most mercurials either bind to cysteine
derivatization. Ones choice must insure that the
sulphurs or histidine nitrogens. In addition to plat-
differences in diffraction amplitudes due to heavy-
inum and mercury, Fig. 6.1 show the many elements
atom contributions are larger than the errors in
successfully used in isomorphous replacement
data measurement. The size of the heavy atom
phasing.
(atomic number) and the number of sites required
for successful phasing are proportional to the size
(molecular weight) of the macromolecule. Larger
macromolecules may require not only atoms of high 6.4 Heavy atoms and their ligands
atomic number, but also more than one heavy atom
The preparation of heavy-atom derivatives and
per molecule. For example the structure determina-
selection of reagents have been extensively reviewed
tion of the ribosome required heavy-atom clusters
+2 (Abdel-Meguid, 1996; Blundell and Johnson, 1976;
such as Ta 6 Br 12 (Ban et al., 2000). Therefore, when
Petsko, 1985; Kim et al., 1985; Holbrook and Kim,
studying larger macromolecules it may be useful
1985; Garman and Murray, 2003). Historically, heavy
to calculate the magnitude of the change in the
atoms have been grouped into class A and class
diffraction signal before deciding which heavy atom
B elements based on their ligand preference. Class
to try. Crick and Magdoff (1956) showed that the
A elements prefer ‘hard’ ligands such as carboxy-
average fractional intensity change (∆I/I) for acen-
lates and other oxygen containing groups. These
tric reflections can be estimated from the following
ligands are electronegative and form electrostatic
equation:
interactions with the derivatives; they include the
1/2 carboxylates of aspartic and glutamic acids and the
∆I/I = (2N H /N P ) (Z H /Z P ) (6)
hydroxyl of serine and threonine. On the other hand,
where N H and N P are the number of heavy atoms class B elements prefer ‘soft’ ligands such are those
and non-hydrogen protein atoms, respectively; Z H containing sulphur, nitrogen, and halides. These
is the atomic number of the heavy atom and include methionine, cysteine, and histidine. The lat-
Z P is 6.7 (the average atomic number of protein ter two amino acids are the most reactive amongst
atoms). all 20 naturally occurring amino acid residues. The
In the case of small proteins, inspection of the cysteine’s sulphur is an excellent nucleophile; it will
amino acid composition can give valuable insights reactirreversiblywithmercuricionsandorganomer-
into which reagents should be tried first. For exam- curials at wide range of pHs. The thiolate anion
ple if the protein contains no free cysteines or also forms stable complexes with class B metals,
histidines it may be best to start soaking with com- but since cysteines are almost totally protonated at
pounds other than mercurials, or to genetically pH 6 or below, this reaction is more sensitive to
engineer heavy-atom binding sites as was done pH than that with mercurials. The imidizole side
with the catalytic domain of γδ resolvase (Abdel- chain of histidine is also highly reactive, particu-
Meguid et al., 1984; Hatfull et al., 1989). However, larly above pH 6 where it is unprotonated. It reacts
assuming a normal distribution of amino acids, one well with reagents containing platinum, mercury,
should begin with platinum compounds such as and gold.
K 2 PtC1 4 (the most widely successful heavy-atom In addition to the amino acids described above,
reagent), which binds mainly to methionine, his- several other amino acid residues are also reac-
tidine, and cysteine residues. Petsko et al. (1978) tive toward compounds containing heavy atoms.
have described the chemistry of this reagent in a These are the side chains of arginine, asparagine,
variety of crystal mother liquors. They also con- glutamine, lysine, tryptophan, and tyrosine. Those
cluded that most other platinum compounds react that are not reactive are alanine, glycine, isoleucine,