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94 MACROMOLECULAR CRYS TALLOGRAPHY

a difference Fourier map. Again the success of this References
feed-back technique depends on the correctness of
Abdel-Meguid, S. S. (1996). Structure determination using
the phasing model.
isomorphous replacement. Method Mol. Biol. 56, 153–171.
Abdel-Meguid, S. S., Grindley, N. D. F., Templeton, N. S.
and Steitz, T. A. (1984). Cleavage of the site-specific
6.8 Refinement of heavy-atom positions recombination protein γδ resolvase: the smaller of the
two fragments binds DNA specifically. Proc. Natl. Acad.
Once the positions of the heavy atoms have been
Sci. USA 81, 2001–2005.
determined, their accuracy can be significantly
Ban, N., Nissen, P., Hansen, J., Moore, P. B. and
improved through least squares refinement. This
Steitz, T. A. (2000). The complete atomic structure of the
is achieved by allowing the heavy-atom positions, large ribosomal subunit at 2.4A resolution, Science 289,
thermal parameters, and occupancy to vary, while 905–920.
minimizing the difference between the structure Blundell, T. L. and Johnson, L. N. (1976). Protein Crystallog-
factor amplitudes calculated from the heavy-atom raphy. Academic Press, London.
model and experimentally measured amplitudes. It Crick, F. H.C. and Magdoff, B. S. (1956). The theory of the
is important to note that thermal parameters and method of isomorphous replacement for protein crystals.
occupancy are often correlated and should not be Acta Crystallogr. 9, 901–908.
Drenth, J. (1999). Principles of Protein X-ray Crystallography,
refined in the same cycle. A good strategy in heavy-
2nd edn. Springer-Verlag, Berlin.
atom refinement is to obtain the best possible heavy-
Drew, H., Takano, T., Takana, S., Itakura, K. and
atom model for each derivative alone, and then add
Dickerson, R. E. (1980). High-salt d(CpGpCpG), a left-
one derivative at a time while refining, to insure that
handed Z’ DNA double helix. Nature (London) 286,
the best derivative does not dominate phasing to the 567–573.
exclusion of others. Also, it is better to omit minor Garman, E. and Murray, J. W. (2003). Heavy-atom deriva-
sites from the initial refinement if there is doubt tization. Acta Crystallogr. D 59, 1903–1913.
about their existence. These sites can be included Green, D. W., Ingram, V. M. and Perutz, M. F. (1954).
in later steps of refinement when it becomes more The structure determination of heamoglobin: IV. Sign
certain that they are real. determination by the isomorphous replacement method.
Proc. R. Soc. London A225, 287–307.
Hatfull, G. F., Sanderson, M. R., Freemont, P. S., Raccuia,
6.9 Conclusion P. R., Grindley, N. D. F. and Steitz, T. A. (1989). Prepa-
ration of heavy-atom derivatives using site-directed
Although the technique of isomorphous replace- mutagenesis: introduction of cysteine residues into γδ
ment was the dominant crystallographic technique resolvase. J. Mol. Biol. 208, 661–667.
for determination of de novo protein structures since Holbrook, S. R. and Kim, S.-H. (1985). Crystallization
its development in the early 1950s to the early and heavy-atom derivatives of polynucleotides. Method
Enzymol. 114, 167–176.
1990s, today it is but one of several techniques
Kendrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G.,
used routinely by those interested in elucidating
Wyckoff, H. W. and Phillips, D. C. (1958). Nature
thethree-dimensionalstructuresofmacromolecules.
181, 662.
Now those interested in crystallographic structure
Kendrew, J. C., Dickerson, R. E., Strandberg, B. E.,
determination can choose from a number of meth- Hart, R. G., Davies, D. R., Phillips, D. C. and Shore, V.
ods to solve the phase problem, including single (1960). Nature 185, 422.
and multiple isomorphous replacement each with Kim, S.-H., Shin, W. C. and Warrant, R. W. (1985). Heavy
or without anomalous scattering, single- and multi- metal ion-nucleic acid interaction. Method Enzymol. 114,
wavelengthanomalousdiffraction(SAD/MAD),and 156–167.
molecular replacement. The choice of which method Patterson, A. L. (1934). A Fourier series method for the
determination of the components of interatomic dis-
to use can depend on a number of factors, such as the
tances in crystals. Phys. Rev. 46, 372–376.
availability of synchrotron radiation, atomic coordi-
Petsko, G. A. (1985). Preparation of isomorphous heavy-
nates of homologous protein, protein expressed in
atom derivatives. Method Enzymol. 114, 147–156.
the presence of selenomethionine, etc.