Figure 7. Electron microscopic analysis of the Nrx1α ectodomain (NX1α_EC).

(A) A raw image of the negatively stained NX1α_EC. Scale bar: 50 nm. (B) Spontaneous degradation of purified NX1α_EC. SDS-PAGE analysis of NX1α_EC protein immediately after purification (left) and again after 2 months storage at 4°C (right) shows that the protein undergoes proteolytic cleavage to produce N-terminal (30 kDa) and C-terminal (110-kDa) fragments. (C) Two-dimensional class averages (upper row images) obtained from multiple electron micrographs and corresponding projection views produced from the 3-D volume map (lower row images). Scale bar, 10 nm. (D) Three-dimensional reconstruction of the NX1α_EC created from multiple oriented particles. (E) Predicted 3D domain organization within the NX1α_EC fragment. Atomic coordinates for NX1α(III) are manually fitted to the densities corresponding to the LNS5-6 and LNS3-4 segments, keeping the C-terminus of LNS4 and the N-terminus of LNS5 close enough to be connected. Domain connectivity was also considered when fitting the LNS2 domain structure (PDB ID: 2H0B) into the density at the bottom. LNS1+EGF1 segment disappeared in the reconstructed volume was not assigned.