Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors

NTF2 and importin-beta are transport factors that mediate nuclear protein import and which interact with nuclear pore proteins (nucleoporins) during translocation from the cytoplasm to the nucleus through nuclear pore complexes. We employed a native gel electrophoresis method to assess the interaction of nucleoporin constructs that contain FxFG sequence repeats with NTF2 and truncation mutants of importin-beta to determine suitable fragments for crystallization. Based on these data, we obtained crystals of complexes between yeast NTF2 and a construct containing five FxFG nucleoporin repeats from the yeast nucleoporin Nsp1p and between a construct containing residues 1-442 of human importin-beta and the same nucleoporin construct. The yeast NTF2-nucleoporin crystals have trigonal symmetry and diffract past 2.8 Angstrom resolution using synchrotron radiation, whereas the importin-beta -nucleoporin complex crystals have P2(1)2(1)2 orthorhombic symmetry and diffract past 3.2 Angstrom resolution. (C) 2000 Academic Press.