Particle identification in protein solution and plain buffer. 3D reconstructions of electron micrographs of a 35.4-µM D(1–4) rat CEACAM1 ectodomain solution and plain buffer were achieved by filtered backprojection and COMET refinement. (A) Histogram displaying the volume distribution (given in voxels) of all structures in a reconstructed volume of D(1–4)-containing specimen identified by SWS (1 voxel = 5.74 × 5.74 × 5.74 Å3). The threshold was 1,000 voxels for D(1–4) dimers and 500 voxels for monomers. All volumes <500 voxels were identified as background noise. All but no additional structures that were identified by SWS were also identified by gray-level thresholding. (B) Comparison of two monomer and two dimer particles from the tomogram analyzed in A identified by SWS (top; surface rendered; volumes given in voxels) and gray-level thresholding (bottom; volume rendered). Bars, 5 nm. (C and D) Comparison of the CEACAM1 D(1–4)-His solution (C) and plain buffer (D), both scaled to the same intensity levels by gray-level thresholding. 30-pixel-deep slices (300 × 160 × 30; pixel size, 5.74 Å) of the total reconstructed volumes are shown. Bars, 50 nm.
