Page 130 - Macromolecular Crystallography

MAD PHASING 119

followed by soaking in HoC1 3 . Although MAD such as argon and krypton have been utilized for this
phasing was not used in this particular structure purpose (Cohen et al., 2001; Quillin and Matthews,
determination, it illustrates the potential for incor- 2003). For larger proteins, clusters of heavy atoms
poration of atomic species which can provide larger have been successfully used (Rudenko et al., 2003).
anomalous signals (Urs, 1999). Other types of spe- Bijvoet (S Bij−Calc ) and dispersive (S Dis−Calc ) signal
cialized techniques include labelled substrates or levels expected from the incorporated anomalously
inhibitors that carry atoms or groups that scatter scattering species can be estimated through use of
anomalously. the following formulae:
The most broadly used, general technique is
1/2 1/2 f
the replacement of methionines in proteins by S Bij−Calc = 1 N A Peak and
selenomethionines. The technique, initially intro- 2 N P Z eff
ducedbyHendricksonandcoworkers(Hendrickson 1/2 1/2 |f − f
1 N A Edge Remote |
et al., 1990), has been widely used. Essentially, S Dis−Calc =
it involves expression of the cloned protein of 2 N P Z eff
interest in a methionine auxotrophic strain with
where
exogenously provided selenomethionine as the sole
source of this amino acid. Several reviews that N A = number of anomalous scatterers
describe the technology and the problems involved N P = number of non-hydrogen atoms in the macro-
(Hendrickson, 1998), including methods for replac- molecule
ing both methionine and cysteine residues by their Z eff = effective scattering factor of an atom (6.7 for
seleno analogs (Strub et al., 2003), and replace- protein atoms).
ment of other residues by methionines to generate
sites for selenium atoms (Gassner and Matthews, 8.3.2 Choice of wavelengths
1999), are available. Although achieving incorpo-
ration of selenomethionines into proteins expressed Generally, data at three wavelengths is preferred for
in non-bacterial cell cultures is still in many cases a determination of both Bijvoet and dispersive differ-
challenge, it has been accomplished in some cases ences, in case of a single anomalous species in the
(Lustbader et al., 1995; Wu et al., 1994). Techniques unit cell. Although in principle, the remote wave-
for introduction of selenium into nucleic acids and length can double as the peak wavelength, as long
oligonucleotides have also been described (Teplova as it is on the short wavelength side of the absorp-
et al., 2002). tion edge (Peterson et al., 1996), use of an additional
A second set of general methods is the classical wavelength improves accuracy and precision of the
approach used for derivatization of crystals in MIR determined phases (Hendrickson, 1991). In particu-
phasing (Garman and Murray, 2003). One of the lar for methods that use least squares calculations in
advantages of MAD methods over MIR is that iso- estimation or refinement of parameters, it provides
morphism is not a requirement. The structure of a valuable additional observations. Bijvoet differences
derivatized protein that is non-isomorphous with are determined by measurements of either Bijvoet
the native can be determined directly from MAD or Friedel mates at the wavelength corresponding to
data, and the native structure subsequently deter- the absorption peak, or on its short wavelength side.
mined through molecular replacement, if necessary. Dispersive differences require the measurement of
Among the more recent developments in techniques intensities at two different wavelengths, one at a
of anomalous scatterer incorporation are those that wavelength close to the inflexion point of the absorp-
involve binding of anomalous scatterers to macro- tion spectrum (Fig. 8.1), and a second one far enough
molecular surfaces using short soaks. Compounds away to enable as a large a signal as practicable. It is
that can be used include both negative ions such important, in many cases, to measure the absorp-
as bromide (Dauter, 2000, 2002) as well as positive tionspectrumfromthecrystalsunderstudysincethe
ions such as rubidium (Korolev et al., 2001). In addi- environment of the anomalous scatterer might sig-
tion, binding cavities in proteins for inert gas atoms nificantly change the position of the infection point