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Figure 1.
Figure 1. Stereo image of the F[o] − F[c] omit electron
density for the bound peptide. The density contoured at 2.7σ is
shown only around peptide residues
Gly^P319-Pro-Gly-Arg-Aib-Phe^P324, which form the S-shaped
double turn in the Fab combining site. When compared to the
density for RP142 [Ghiara et al 1994], additional electron
density is seen around the extra methyl group of the Aib
residue. Peptide residue numbers are according to the BH10
isolate sequence [Ratner et al 1985], and are preceded by the
letter P. Peptide Aib142 was synthesized on a methyl
benzhydrylamine (MBHA) resin following standard procedure
[Schnolzer et al 1992]. The Aib was coupled manually for 30
minutes followed by a second coupling for 60 minutes. Following
treatment with anhydrous HF and purification by preparative
HPLC, the peptide was characterized by analytical HPLC and
electrospray mass spectrometry (observed 2891.6 (+/−0.6)Da;
calculated average 2891.5 Da). Fab fragments were prepared by
enzymatic digestion of monoclonal antibody 59.1 (IgG1) with
pepsin followed by controlled reduction in the presence of
cysteine. Crystals of Fab 59.1 in complex with peptide Aib142
were obtained by vapor diffusion in sitting drops (2 to 5 μl),
that contained between 1.4 and 1.8 M mixed phosphate
(NaH[2]PO[4] and K[2]HPO[4] pH range 5.0 to 6.75), with 18 mg/ml
Fab at 22°C and a tenfold molar excess of Aib142. The
crystals resemble the Fab59.1-RP142 crystals in morphology.
Screenless precession photographs confirmed that the cell was
orthorhombic, with similar cell dimensions to the Fab59.1-RP142
complex (a = 89.7 Å, b = 154.0 Å, and c = 121.9
Å [Ghiara et al 1994]). X-ray diffraction data were
collected from a large crystal (0.8 mm × 0.4 mm ×
0.3 mm) grown in 1.4 M mixed phosphate buffer, pH 6.5, on a MAR
image plate mounted on a Siemens X-ray generator at a maximum
power of 100 mA and 50 kV. Detector data were processed with
MOSFLM [Leslie et al 1986]. The space group was confirmed to be
C 222[1], with unit cell dimensions a = 89.9 Å, b = 154.4
Å, and c = 121.4 Å. The final data set consists of
50,028 total observations of 19,192 unique reflections and is
92% complete to 2.8 Å (90% complete in the 2.8 to 2.9
Å outer shell) with an R[sym](I) value of 8.0%. The
Fab59.1-Aib142 structure was determined by molecular
replacement. As the crystal cell dimensions for the two
complexes (with RP142 and Aib142) are within 0.5 Å of each
other and their respective peptides differ only at one residue,
the refined Fab59.1-RP142 complex structure ([Ghiara et al 1994]
Brookhaven Protein Data Bank, code 1ACY) was used as the initial
starting model. After rigid body refinement using X-PLOR
[Brunger 1992], the R-value was 0.24 for 8.0 to 4.0 Å data
with F>2σ. The model was then refined with the positional and
simulated annealing protocols in X-PLOR to an R-value of 0.27
for all data from 12.0 to 2.8 Å. The RP142 peptide was
included in the model at this stage to prevent any side-chains
from CDR loops from moving into the peptide electron density
during simulated annealing. F[o] − F[c] omit maps, calculated
by excluding the peptide, clearly demonstrated no significant
difference for the Aib142 peptide conformation. One cycle of
model building of the entire complex into 10% 2F[o] − F[c]
omit maps was then undertaken with FRODO [Jones 1978 and Jones
1982]. The hydrogen atom on Ala of the peptide was replaced with
a methyl group with Insight II, version 2.2 (Biosym
Technologies, 1993), to incorporate the Aib residue into the
model; the parameter and topology files used in X-PLOR [Engh and
Huber 1991] were also modified to include specifications for the
Aib residue. Two more cycles of model building and refinement
resulted in the current structure, with an R-value of 0.22 for
all data in the 12.0 to 2.8 Å range, with atomic B-factor
refinement (the average overall B-value is 30 Å^2) and rms
deviations from ideality for bond lengths and angles of 0.015
Å and 2.0°, respectively. Coordinates have been
deposited in the Brookhaven Data Bank, code 1A T 1.
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