Page 98 - Macromolecular Crystallography

CHAPTER 6

Solving the phase problem using

isomorphous replacement

Sherin S. Abdel-Meguid

6.1 Introduction The addition of one or more heavy atoms to a macro-
molecule introduces differences in the diffraction
From the mid 1950s, when it was first introduced,
pattern of the derivative relative to that of the native.
till the mid 1990s, the method of isomorphous
If this addition is truly isomorphous, these differ-
replacement played a central role in the determi-
ences will represent the contribution from the heavy
nation of almost all unique macromoleular struc-
atoms only; thus the problem of determining atomic
tures. Isomorphous replacement was the technique
positions is initially reduced to locating the position
used in the first successful high-resolution struc-
of a few heavy atoms. Once the positions of these
ture determination of a protein molecule, myoglobin
atoms are accurately determined, they are used to
(Kendrew et al., 1958; Kendrew et al., 1960). It
calculate a set of phases for data measured from
was developed by Perutz and coworkers in 1954
the native crystal. Although, theoretically, one needs
(Green et al., 1954) while working on the structure
only two isomorphous derivatives to determine the
determination of haemoglobin. The technique was
three-dimensional structure of a biological macro-
introduced to solve the ‘Phase Problem’, the loss
molecule, in practice more than two are needed.
during X-ray diffraction data measurement of the
This is due to errors in data measurement and scal-
relative phase shifts associated with each diffrac-
ing and in heavy-atom positions, as well as lack of
tion point (maximum). Although the amplitude of a
isomorphism.
diffraction maximum can be directly measured from
The search for isomorphous derivatives is as
diffractingcrystalsbycountingphotonsorrecording
empirical as searching for crystallization conditions.
intensities, phasesareindirectlydeterminedbecause
Numerous heavy atoms must be screened before
there are no lenses that can bend and focus X-rays.
finding the one or more that binds to the pro-
Thus, the isomorphous replacement method was
tein without damaging the crystal. The soaking
developed to computationally calculate phases from
of native crystals in a solution containing heavy
the intensities of the diffracted waves.
atoms gives rise to one of four outcomes. The best
The technique of isomorphous replacement
outcome would be an isomorphous heavy-atom
requires the introduction of atoms of high atomic
derivative containing one or a small number of
number (heavy atoms; Fig. 6.1) into the macro-
heavy atoms identically attached to each protein
molecule under study without changing the crystal’s
molecule in the crystal, resulting in distinct changes
unit-cell parameters or orientation of the protein in
between the diffraction patterns obtained from
the cell (Abdel-Meguid, 1996). This is commonly
derivative and native crystals. At the other extreme,
done by soaking native crystals in a solution con-
the soaking process results in no such detectable
taining the desired heavy atom. The binding of these
changes when comparing native and derivative
atoms to the functional groups of macromolecules is
crystals. The two remaining outcomes are either
facilitated by the presence of large liquid channels
the native crystal gets destroyed during soaking or
in crystals, in which the functional groups protrude.
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