Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

308 Boyd et al.
Fig. 3. Histogram of 1 D NH values measured for the LDLR-AB cbEGF domain pair.
4. Values for D a and R are obtained by least squares minimization of Eq. 4 and
using the estimates of D xx, D yy, and D zz found for LDLR-AB yields values of
23.4 Hz and 0.17, respectively.
3.5. Structure Refinement
1. A modified simulated annealing protocol incorporating residual dipolar couplingderived
restraints has been described by Tjandra et al. (7) and may be obtained
from the authors. In this method, each N–H bond vector is constrained relative to
a molecular axis system with axial and rhombic components D a and R. Input data
for the procedure comprise estimates for D a and R, as well as a table of restraints
in the format shown in Fig. 4. Each restraint relates the residual dipolar coupling
of residue n to the orientation of the molecular axis system, defined here as residue
500. The molecular axis system is contained in a separate PDB file. It contains
four atoms with coordinates corresonding to x, y, z, and the origin. The origin of
the molecular axis system is fixed, and its orientation is allowed to float during the
simulated annealing protocol, such that it adopts an alignment in best agreement
with the experimental measurements. In order to avoid steric clashes, the origin of
the axis system should be localized far away (≥50 Å) from the protein.
2. Agreement with experimentally derived restraints is achieved via minimization
the term E dipolar (7):