Figure 3. HDX‐MS and 3D variability analysis highlight dynamic regions in KCCs.

• A
  HDX‐MS difference plot illustrating areas with altered deuterium exchange rates for PKO and PM of KCC3b. Relative fractional uptake differences (PKO minus PM Mutant) at three indicated time points are plotted against amino acid range covered by peptides. Negative uptake difference indicates an increased hydrogen exchange of PM. Positive uptake difference indicates a decreased hydrogen exchange of PM. All data points are mean from triplicates. Grey highlighted region indicates peptides with increased hydrogen exchange of the PM variant for an N‐terminal portion not resolved in the cryo‐EM structure.
• B, C
  Conformational dynamics of full‐length KCC1Δ19 and KCC3b‐PM structures inferred from normal modes (3D variability analysis. Movements of flexible regions are indicated by arrows. Extent of movement is quantified in Å or ° for KCC1Δ19 (values in blue or red) and KCC3b‐PM (values in dark blue or dark red).
• D, E
  Co‐existing alternative conformations of the CTD outer lobe helices α8 in KCC1Δ19 (D, violet and purple helices) and α7 in KCC3b‐PM (E, blue and orange helices). Cα atom locations of T629 within the KCC1Δ19 α8 helices are highlighted in yellow. Alternative conformations of α10b of KCC1Δ19 are highlighted in teal and light teal cartoon representation.
• F
  Cartoon representation of KCC3b‐WT CTD (pdb: 6MIY) with alternative α8 and α10b conformations, highlighted in violet and teal, respectively. Phosphorylated residue T940‐P is shown as yellow sticks.
• G
  Cartoon representation of the CTD of CeKCC1 (pdb: 6VWA) with alternative α8 and α10b conformations, highlighted in purple and light teal, respectively.
• H
  Cartoon representation of the CTD of DrNKCC1 (pdb: 6NPJ).

Data information: Inner lobes of the CTDs in panels (D‐H) are shown in light grey cartoon representation, outer lobes are highlighted in colours.