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Figure 1.
Fig. 1. (a) A cartoon of the structure of the domain-swapped
dimer of NCAM2 Ig1. Hinge region residues Leu7-Ser8 and other
residues (Val10 and F19) that participate in stabilization of
the dimer are shown as red sticks. The NCAM2 Ig1 β-strands are
labelled (A, A', B, C, C', D, E, F, and G) according to the
nomenclature used for NCAM Ig1.^5 Strand C' is in a perturbed
β-strand conformation. (b) A stereo view of the strand-exchange
region of the NCAM2 dimer. Omit-map electron density is shown as
chickenwire (grey) around the swapped β-strand. The hydrophobic
cluster of residues assumed to facilitate domain swapping and to
stabilize dimer formation is shown as red sticks. The human Ig1
domain of NCAM2 used in this study was prepared with a
C-terminal His tag using PCR amplified cDNA (Ensembl Gene ID
ENSG00000154654; RZPD, Germany) for sub-cloning into the
ClaI/NotI sites of the pPICZα C plasmid (Invitrogen). The
protein consists of two N-terminal residues (Ser and Met)
remaining from the cloning procedure, followed by the amino
acids (19–114) of human NCAM2 (Swiss-Prot code O15394) and six
C-terminal histidine residues. The first four amino acids (SMAL)
and the last seven amino acids (KHHHHHH) are not defined by
electron density due to flexibility. The construct was verified
by DNA sequencing. The recombinant plasmid was linearized with
SacI enzyme and used for transformation of Pichia pastoris
strain KM71H (Invitrogen). Transformation and selection were
performed by the protocol supplied by the manufacturer. A
pre-induction culture was grown for 48 h in BMGH medium before
transfer and continued growth and induction in BMMH medium for
24 h. The secreted protein was purified using Ni-NTA (Qiagen)
affinity chromatography followed by gel-filtration
chromatography in phosphate-buffered saline (PBS) on a Superdex
75 column (GE Healthcare). Fractions corresponding to the
monomeric form of the protein were collected and stored for two
days at 4 °C, and the protein was subsequently used for
crystallization experiments. All figures were prepared using the
program PyMOL (http://www.pymol.org).
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