The EcppnP act as a non-canonical dimerization mode with a conserved dimer interface
We then analyzed the symmetric unit and found that the Ec ppnP adopt a pseudodimer conformation (Figure 1C ). In the homodimer of Ec ppnP, the interactions between each subunit are mediated mainly by the anti-paralleled 6-stranded β-sheet (β1-β2-β3-β10-β5-β8), herein termed the inner sheet. The anti-paralleled 5-stranded β-sheet (β7-β6-β9-β4-β11) is located outside of the dimer and termed the outer sheet (Figure 1C ). The interactions between the inner sheets are mostly mediated by the four β-strands β1-β2-β3-β10 and are largely mediated by hydrophobic interactions. Residues involved in the dimer formation are symmetric in the Ec ppnP structure including Leu2, Ile15, Phe17, Val27, Val29, Val31, and Pro86 (Figure 1D ). These residues mainly located in β2-β3 composed a hydrophobic surface region for the dimer formation. Size exclusion chromatography (SEC) is a commonly used approach for the identification of a protein oligomeric state. According to our SEC results, the wildtype Ec ppnP protein is present as a dimer in the solution same as we observed in the crystal (Figure 2A ). And then, we select to mutate the Val27 and Val29 residues to aspartate (V27D/V29D) and test the in-solution status ofEc ppnP with SEC. However, the mutate Ec ppnP is not stable enough and quickly degraded (Figure 2B ).
To further validate this dimer status of Ec ppnP, we solved another three ppnP structures in different species includingPseudomonas aeruginosa (strain PA0750) Pa ppnP,Vibrio cholerae (strain SO5Y) Vc ppnP, Salmonella enterica (subsp. enterica serovar Typhimurium strain SL7207)St ppnP. The diffraction data of Pa ppnP, Vc ppnP, andSt ppnP were also determined with high resolution to 1.50 Å, 1.4 Å, and 1.2 Å, respectively (Table 1 ). Interestingly, all of the three structures present dimer conformation in one asymmetric unit (Figure 2C, 2D, 2E ). We then superimposed each structure of them to Ec ppnP, the superposition results showed that all the three structures have minimal RMSD to Ec ppnP, with 0.446 Å ofPa ppnP to Ec ppnP, 0.476 Å of St ppnP toEc ppnP and 0.406 Å of Vc ppnP to Ec ppnP. Therefore, the homodimer conformation of Ec ppnP is well-conserved in other species both from the quaternary structure (Figure 2C, 2D, 2E ).