Abstract
As an example of structure determination in the 3.5-4.5 A resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3 A resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at approximately 4 A resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible.
Highlights
As more and more challenging and complex systems are being studied, X-ray crystallography is increasingly hampered by weakly diffracting crystals
The structures of some of the components may be known at high resolution, while those of others are unknown. Solving such complexes should be possible as the determinancy point, that is the Bragg spacing limit at which the number of independent diffraction intensities equals the number of flexible torsion angles, is generally in the range between 4.9 and 6.4 A ; for example, for a crystal structure with 50% solvent content the determinancy point is 5.4 A (Hendrickson, 2009)
Try B-sharpening electron-density maps paper, we describe our experiences with the structure solution and refinement of a difficult low-resolution crystal structure, the ATPase p97/VCP
Summary
As more and more challenging and complex systems are being studied, X-ray crystallography is increasingly hampered by weakly diffracting crystals. We obtained low-resolution diffraction data sets of p97/VCP in three nucleotide states (ATP at 3.5 Aresolution, ATPÁAlFx at 4.5 Aresolution and ADP at 4.25 Aresolution; DeLaBarre & Brunger, 2003, 2005) and employed molecular replacement using the known structure of the N-D1 fragment (PDB code 1e32) as the search model; the structure of the isolated D2 domain was unknown at the time. The revised full-length models showed significant improvement in both model geometry and R values compared with the original structures (Davies et al, 2008) We refer to those models as the re-refined p97/VCP structures (PDB codes 3cf1, 3cf and 3cf).
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