The formation of single, well-diffracting crystals is a requirement for any
molecular structure determination by x-ray crystallography. Crystallizatio
n of biological macromolecules can represent a significant obstacle when th
e subject exhibits internal flexibility or indiscriminate self-association.
In such cases, the removal of inherently flexible regions and the addition
of stabilizing ligands can improve the probability of crystal formation an
d ordered growth. We have applied these principles in order to form crystal
s of the Rel homology region of transcription factor NF-kappaB in complex w
ith its inhibitors I kappaB alpha and I kappaB beta. None of these molecule
s crystallizes in the absence of a binding partner. Recombinant overexpress
ion of truncated I kappaB alpha required selection of the correct start sit
e. NF-kappaB .I kappaB alpha complex crystals formed under relatively strin
gent conditions. NF-kappaB .I kappaB beta complex crystals were formed by a
nalogy to NF-kappaB .I kappaB alpha, although some modifications in purific
ation and complex formation were necessary due to differences between the i
nhibitors.