Fig. 4: CryoEM and MS characterisation of C4b binding to IgG3. | Nature Communications

Fig. 4: CryoEM and MS characterisation of C4b binding to IgG3.

From: Complement is activated by elevated IgG3 hexameric platforms and deposits C4b onto distinct antibody domains

Fig. 4

a Density corresponding to C4b (orange) reveals a tilted molecule bound to IgG3 Fab domains (green). gC1q are displayed in blue, C1s is displayed in pink and the liposome surface is displayed in grey. b Example of a Coomassie-stained gel showing purified IgG3 (green arrow), C4b (orange arrow), and mixtures with liposomes (Lipos) and human serum (NHS). A new band appeared upon mixture of all required components (blue arrow). Gel was repeated three times. c Model of C4b bound to IgG3 showing sites for covalent attachment as determined using MS. Sites of attachment are indicated with the following colours: STSGGTAALGCLVK, pink; SCDTPPPCPR, cyan; VSNKALPAPIEK, red. Residues identified by MS that attach to C4b are denoted as spheres. The C4b-TED reactive glutamine residue is shown as a blue sphere. d MS analysis of tryptic peptides of C4b (GCGEQTmIYLAPTLAASR, orange; m, oxidised methionine) crosslinked to IgG1 (upper, red) and IgG3 (lower, green). For both samples, extracted ion chromatograms show traces for the C4b peptide (orange) attached to VSNKALPAPIEK (32 min). Only IgG3 has C4b conjugated to other sites. Crosslinked residues are denoted in bold and underlined in the colour corresponding to (c).

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