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Figure 1.
Figure 1. (a) An overlay of the backbone atoms of the 25
lowest-energy NMR structures. The coding sequence for residues
357-425 of SPTREMBL O75400 corresponding to the FF domain was
amplified by PCR from IMAGE cDNA clone 731611 (obtained from the
MRC HGMP Resource Centre) by standard methods and cloned into a
pRSET-derived pHisGro vector. This was used to over-express a
soluble histidine-tagged GroEL apical domain/FF domain fusion
protein in Escherichia coli. The fusion protein was purified
under native conditions using NTA agarose (Qiagen). After
cleavage with thrombin, the FF domain was purified using
ion-exchange chromatography and gel-filtration. The samples for
NMR spectroscopy typically contained 2.5 mM human FF domain in
90% H[2]O/10% 2H[2]O containing 50 mM KCl, 50 mM potassium
phosphate (pH 6.0) at 298 K. The NMR spectra were assigned using
standard NMR methods.[18. and 19.] The assignments have been
deposited in the BioMagResBank under accession numbers PDB 1H40
BMRDB 5537. A set of distance constraints were derived from a
series of NOESY spectra recorded in H[2]O and 2H[2]O with mixing
times of 150 ms. The NOESY spectrum was integrated according to
the cross-peak strengths and calibrated by comparison with NOE
connectivities obtained for standard inter-residue distances
within an a helix. After calibration, the NOE constraints were
classified into the following categories: strong, medium, weak
and very weak, corresponding to inter-proton distance
constraints of 1.8-2.8 Å, 1.8-3.5 Å, 1.8-4.75
Å, and 2.5-6.0 Å, respectively. Hydrogen bond
constraints were included for a number of backbone NH groups
whose signals were observed in a 2D 1H-15N-HSQC recorded in
99.996% 2H[2]O at 298 K (pH 5.0). For hydrogen bond partners,
two distance constraints were used where the distance (D)H-O(A)
corresponded to 1.5-2.5 Å and (D)N-O(A) to 2.5-3.5
Å. Torsional angle constraints were obtained from an
analysis of C', N, C^a Ha and C^b chemical shifts using the
program TALOS.[20.] The three-dimensional structure of the FF
domain was calculated using a dynamic simulated annealing
protocol based upon the work of Nilges et al.[21.] in the
program XPLOR (Brünger, A. T. (1992). X-PLOR Version 3.1: a
system for cystallography and NMR, Yale University, New Haven,
CT). The coordinates have been deposited in the protein
structure database, entry. (b) A ribbon representation of the
lowest-energy structure prepared using the program
MOLSCRIPT.[22.] (c) A ribbon representation of the C-terminal
region of human phosphatase 2C alpha prepared using the program
MOLSCRIPT. [22.] Note there is a break in the electron density
in the loop between the first and second helices.
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