The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Klaile, Esther; Vorontsova, Olga; Sigmundsson, Kristmundur; Müller, Mario M.; Singer, Bernhard B.; Öfverstedt, Lars-Göran; Svensson, Stina; Skoglund, Ulf; Öbrink, Björn

CEACAM1 D(1-4) ectodomain that was polydisperse as a result of varying degrees of glycosylation had an average molecular mass of 81 kD (Fig. S2) , which corresponds to a volume of 580 voxels (1 voxel = 5.74 × 5.74 × 5.74 Å 3 ). All CEACAM1 ectodomains should be found among particles having a volume >500 voxels. With this constraint, gray-level thresholding and SWS returned the same particles, corresponding to CEACAM1 monomers and dimers (Fig. 2, A and B ). In addition to these protein molecular particles, a large number of smaller background structures with volumes up to 500 voxels were recorded by both approaches (Fig. 2 , A and C). Similar amounts of particles of the same size distribution were seen in plain buffer specimens (Fig. 2 D) . Because the reactions occur between both the analyte molecules in the soluble bulk phase and between the soluble analyte and the immobilized ligand. Therefore, we had to design novel algorithms based on a set of plausible homophilic binding reactions that reflect such a case. Three different reaction schemes were developed: