Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Protein Structure Calculation from NMR 275
6. Perform 200 TAD steps at zero reference temperature.
7. Perform 1000 conjugate gradient minimization steps including all restraints.
During the TAD calculation, the list of van der Waals lower distance bounds is
updated every 50 steps, or, during minimization, whenever a torsion angle has
changed by more than 10° since the last update, or after 100 minimization steps.
DYANA is an integrated program, which includes CALIBA (calibration of
NOE intensity vs distance restraints) and a versatile multidimensional gridsearch
algorithm. CALIBA calibrates NOE intensities into distance restraints.
It has different calibration functions for backbone, side-chains and methyl
groups. The calibration functions are V =A/r 6 , V=B/r 4 , V=C/r 4 where V is
the peak volume and r is the corresponding distance. The parameters A, B, and
C are either user-defined or calculated automatically (typically, B = A/d min 2 and
C = B/3, dmin = 2.4 Å). The multidimensional gridsearch algorithm analyzes the
local conformation of an arbitrary molecular fragment of a protein involving the
three torsional angles φ, ψ, and χ 1 of an amino acid residue, determines the
stereospecific assignments of β-protons and generates torsion angle restraints.
4. Notes
1. NOEs are the essential NMR data for defining the secondary and tertiary structures
of a protein because they permit connection of pairs of hydrogen atoms in
amino acid residues that may be far apart in the protein sequence, but close in
space (less than about 5 Å apart). The NOE arises from the transfer of magnetization
between spins coupled by the dipole–dipole interaction in a molecule undergoing
Brownian motion in a liquid (30–32). The intensity of an NOE, i.e., the
volume of the corresponding cross peak in a NOESY spectrum (31,33,34), is
related to the distance r between the two interacting spins by
V < r –6 > f(τc) where r –6 is averaged since the distance r may vary in molecules with inherent
flexibility. The remaining dependence of the magnetization transfer on motion
enters through the function f (τ c) that includes effects of global and internal
motions of the molecule.
The NOE is quantified by the volume or intensity of the corresponding cross
peak in the NOESY spectrum (35). Because the linewidths can vary appreciably
for different resonances, cross peaks should be quantified by integration over the
peak area rather than by measuring peak heights.
2. Measured crosspeak (NOE) volumes are translated to distance ranges. The lower
bound is determined from the sum of the van der Waals’ radii and the upper bound
from the NOE intensity. NOEs are usually translated into upper bounds on interatomic
distances rather than precise distance restraints because the presence of
internal motions, spin diffusion and, possibly, chemical exchange may affect the
intensity of an NOE (35).