Extended Data Fig. 6: Localization of three Ca²⁺ binding sites in PDIA6.

a, Overlay of 2D [¹³C,¹H]-HMQC spectra of 100 μM methyl-labelled PDIA6 in absence (black) and presence of 10 mM Ca²⁺ (purple). Full spectra shown in Supplementary Fig. 3a. b, Overlay of 2D [¹³C,¹H]-HMQC spectra of 100 μM methyl-labelled PDIA6 in absence (black) and presence of 0.05 eq. Gd³⁺ (purple). Full spectra shown in Supplementary Fig. 3b. c, PRE-effect of 0.05 eq. Gd³⁺ on full-length PDIA6 (top) and truncated ΔC PDIA6 (middle) and outcompetition of 0.5 eq. Gd³⁺ by 1 mM Ca²⁺ on full-length PDIA6 (bottom). Residues with substantial loss in intensity are highlighted in shades of purple (I, II and III). d,e, Zoom on Ca²⁺ binding site of calsequestrin low Ca²⁺ form (PDB: 5kn3, yellow) (left) and overlay with PDIA6 (grey) Ca²⁺ binding sites I (d) and II (e) (right). Side chains of Ca²⁺ binding residues shown as sticks. Methyl groups with substantial loss in intensity in Fig. 2g represented as spheres as in Fig. 2h. The Ca²⁺ ions in calsequestrin are coordinated by the amino acid residues Asp/Glu/Asp for site I and Asn/Asn/Asp/Asp for site II, while in PDIA6 by Asp/Ser/Ser and Asn/Glu/Gln/Thr, respectively, resulting in weaker Ca²⁺ binding. f, Overlayed sections of 2D [¹⁵N,¹H]-HSQC spectra of 100 μM PDIA6 domain ab in absence (black) and in presence of excess Ca²⁺ showing assigned residues located at Ca²⁺ binding sites. Full spectra in Supplementary Fig. 4. g, Soluble expression yields of PDIA6 constructs with mutations (S263A, D271L, E280M, N284K, T337V or Q342K) targeting the Ca²⁺ binding sites. h, Fixed HeLa cells transfected with PDIA6–GFP ΔC. Scale bar, 10 μm. i, In vitro phase separation of Dylight 488–PDIA6 EQDN, which has been reported to not bind Ca²⁺ at the C-terminal tail²³. Scale bar, 10 μm.