The aligned sequences of the βSBD, including human HSP70s: BiP (GRP78 or HSPA5), HSP70 (HSPA1A or HSP72), HSPA2, HSC70 (HSPA8 or HSP73), HSPA9 (GRP75, Mortalin-2 or MTHSP70), and HSPA6; Hsp70s from eukaryotes: Stratiomys singularior (UniProt: B2CKI9), Plasmodiophora brassicae (UniProt: A0A0G4IKJ2) and Monodelphis domestica (UniProt: F6VP59); and bacterial Hsp70s: HscA and DnaK from Escherichia coli. Secondary structure elements are indicated above the protein sequence. The highly conserved residues are coloured in red, less conserved in orange, non-conserved in black. The positions of ‘soft mutations’ used in this study are indicated by green asterisks; the AMPylation site (Thr 518) is highlighted by a magenta asterisk.