Fig. 3: Structure of F(ab’)2 in subunit 1 and its flexibility.
a One of five conformations (conformation 4) classified from 3-D variability analysis (3DVA). Threshold of the map is 2.4. b Map obtained by focused-refinement of the conformation 4 at subunit 1 (dotted box in (a)). B factor used for map sharpening is −400 Å2 and the threshold of the map is 2. The two black arrowheads point at the N-linked glycosylation at N332 in Cμ2 dimer. c Zoomed-in view of Fab-1 (green dotted box in (b)), with location of oligosaccharide at N165 on heavy chain indicated by the black arrowhead. Models docked in (b, c) are pdb id 6KXS (cyan, subunit 1 only), pdb id 4JVU (red) and two crystal models of IgM Fab (green and blue, pdb id 2AGJ) for Fab-1 and Fab’−1. The heavy chains are dark green/blue and light chains are light green/blue. The maps for the other four conformations and corresponding focused refined maps are in Supplementary Fig. 5. d Schematic of the heavy and light chain arrangement in F(ab’)2. e Overlay of the models of five maps with different F(ab’)2 conformations aligned at their common Cμ4-Cμ3 core region. The in-plane (\({{{{{\rm{\alpha }}}}}}\)) and out-of-plane (\({{{{{\rm{\beta }}}}}}\)) and the positive/negative axes of F(ab’)2 are defined. The orientation of each Fab/Fab’ arm is defined as the direction from the C-terminus of Cμ2-1 to the centre of the constant domain \({{{{{\rm{\alpha }}}}}}=0^\circ\) is defined as the two-fold symmetry axis of Cμ4-1 A/1B dimer, and \({{{{{\rm{\beta }}}}}}=0^\circ\) is the platform formed by Cμ4-Cμ3 core (black dotted lines). \({{{{{\rm{\alpha }}}}}}\) and \({{{{{\rm{\beta }}}}}}\) are the relative angles between Fab/Fab’ and \({{{{{\rm{\alpha }}}}}}=0^\circ\)/\({{{{{\rm{\beta }}}}}}=0^\circ\). f Summary of \({{{{{\rm{\alpha }}}}}}\) and \({{{{{\rm{\beta }}}}}}\) in the five conformations. The datapoints are the same colours as the maps in (e). The grey datapoints are the Fab/Fab’ values measured from the 20 frames in the volume series calculated by 3DVA showing the continuous flexibility of F(ab’)2 in subunit 1 (Supplementary Movie 1 and Supplementary Movie 2). g Superimposition of the F(ab’)2 structures in all five conformations.
