Page 174 - Macromolecular Crystallography

MODEL BUILDING, REFINEMENT, AND VALIDATION 163

the ‘rippling’ effect caused by Fourier transforms by the ARP/wARP autobuilding module for con-
with incomplete datasets, on top of depriving the struction of a polypeptide chain that is in agreement
optimizer algorithm of the refinement program from with the electron density. In the regions where it
valuable diffraction data. is not possible to make sense of the electron den-
sity features in terms of a protein model, free atoms
are kept. This results in what is called a hybrid
11.2.5 Model building and refinement are
both phase improvement procedures model, a mixture of free atoms and fragments of
the protein structure. Much in the same way as in
ARP/wARP challenged the common separation of the ‘traditional’ X-ray structure solution, the hybrid
model building and refinement by taking a more model undergoes a refinement procedure with the
general view of the underlying phase optimization highly successful maximum likelihood based refine-
through coordinate manipulation and by allowing ment program REFMAC5 (Murshudov et al., 1997)
the extension of the macromolecular model as part in which the parameters of the model are adjusted
of the whole process (Perrakis et al., 1999). This to best fit the experimental data and stereochemical
placed structure solution on a more unified foun- expectations. If the quality of the model is suffi-
dation and encouraged automation in linking the ciently high, the phases improve overall and result
entire procedure. in an enhanced electron density into which a more
Model building is an interpretation of the cur- accurate and more complete model may be built
rently available electron density. Refinement is the at the next cycle. ARP/wARP, like human crystal-
adjustment of the built model to fit better to the lographers, links model building and refinement
experimental data. A crucial point here is that a together into a unified process that iteratively pro-
density map computed from the refined model is ceeds towards a more complete macromolecular
generally better than the map obtained from the model.
same model before the refinement. This then allows
for an even better model to be built. Thus, refinement
is needed to improve the outcome of model build- 11.3 Software packages
ing by generating a better electron density map and 11.3.1 An overview of refinement programs
model building is needed to provide a model in the
first place and to provide stereochemical restraints Refinement is an optimization problem. Therefore
for the subsequent refinement to proceed smoothly. we choose to outline the current programs based
This viewpoint merges these two steps into one on how the optimization problem is formulated
model optimization process. (including the variables and the objective function)
The ARP/wARP procedure is largely based on and the method of choice for locating the min-
the concept of describing the electron density with ima (the solution method). The parameterization
a collection of free atoms, originally introduced for of a macromolecular model is a crucial step and
phase extension by Isaacs and Agarwal (1985). The should reflect the amount of available data that
main a priori knowledge in this case is the fact that can be used to optimize the model parameters.
proteins consist of atoms, although this informa- This fluid parameterization is typically replaced by
tion alone is hardly sufficient.ARP/wARP, however, standard atomic coordinates (xyz), a displacement
takes this further – before linking the atoms, one parameter (B), and occupancy (occ) parameteriza-
has to position them correctly, not necessarily into tion in which the amount of data is accounted
peaks of the density but preserving at least the for in the weights (tightness) of the stereochemical
first order stereochemical information – the inter- restraints. Other parameterization approaches use
atomic distances. Free atoms themselves account torsion angle, rigid body, and NCS constrained/
for the electron density features without the need restrained (hard/soft) parameterization. Additional
to apply additional chemical knowledge or distance parameters include those used for modelling the
restraints between them. The coordinates of the free bulk solvent. Within the free atoms framework, the
atoms are refined, updated where needed, and used atom type itself enters as a parameter since such