Supplemental Information
Cloning, expression and purificationof EcRraA
The RraA gene was PCR-amplified from E coli K12 genomic DNA using PrimeSTARTM HS DNA polymerase(TaKaRa). After amplification of the target gene, the resulting fragmentencodingfull length residues of EcRraA was digested with NdeI and XhoI and cloned into the vector pET28a.EcRraA protein was expressed in the host strainE. coli BL21 (DE3). Expression was performed in 2.0 Lof Luria-Bertani medium, which was incubated at 310 K until the OD600 reachedabout 0.6. EcRraA expression was induced by the addition of 0.15 mMIPTG for an additional 4 hrs at310K. For the preparation of soluble protein fractions, cells from the 2.0 Lculture were first pelleted and then resuspended in 50 ml cold lysisbuffer (20 mM Tris pH 8.0 and300 mM NaCl). It was then homogenizedby sonication on ice. The clearsupernatant containing soluble proteinwas collected by centrifugation.
EcRraA was purified by the same methods for RraA from P. aeruginosa in this report.
Oligomeric state determination of EcRraA
Size exclusion chromatography was performed with a Superdex 200 column (GE Healthcare) by the same method for RraA from P. aeruginosa in this report. The protein sample or molecular mass standards were applied to the Superdex 200 column and eluted with 50mM Tris-HCl pH8.0 and 200mM NaCl. Standard proteins were: aldolase(158kDa), conalbumin(75kDa), ovalbumin (43kDa),chymotrypsinogen A (25.6kDa), ribonuclease (13.7kDa) and the void volume were determined with Blue Dextran (GE Healthcare). The peak elution volumes (from flow measured at 280 nm) were used for the calculation of the standard curve equation (Log MW=-0.03689Ve+7.72641, with the R2 value of 0.9634). EcRraA was eluted as a single peak at volume 75.45 ml corresponding to a molecular weight of 87.7 kDa.
The hydrogen bonds for assembling RraA trimer
Interaction Between A-BMonomer A / Monomer B
Residues / Atom / Residues / Atom
Asn148 / ND2 / Asp100 / OD1
Asn148 / O / Ser114 / OG
Asn149 / ND2 / Ser114 / OG
Asn149 / O / Ser114 / OG
Asp10 / OD1 / Arg97 / NH2
Asp10 / OD2 / Arg97 / NH2
Val19 / O / His115 / NE2
Interaction Between B-C
Monomer B / Monomer C
Residues / Atom / Residues / Atom
Asn148 / ND2 / Asp100 / OD1
Asn148 / O / Ser114 / OG
Asn149 / ND2 / Ser114 / OG
Asn149 / O / Ser114 / OG
Asp10 / OD1 / Arg97 / NH2
Asp10 / OD2 / Arg97 / NH2
Val19 / O / His115 / NE2
Interaction Between A-C
Monomer C / Monomer A
Residues / Atom / Residues / Atom
Asn148 / ND2 / Asp100 / OD1
Asn148 / O / Ser114 / OG
Asn149 / ND2 / Ser114 / OG
Asn149 / O / Ser114 / OG
Asp10 / OD1 / Arg97 / NH2
Asp10 / OD2 / Arg97 / NH2
Val19 / O / His115 / NE2
Supplemental figure legends
Figure S1.Size exclusion gel filtration chromatography of the RraA from P. aeruginosa using a Superdex 200 column,the RraA protein was eluted as a single peak at volume 73.45 ml corresponding to a molecular weight of 103.9 kDa.
Figure S2. RraA from P. aeruginosa dynamic light-scattering experiment using a DynaPro MS/X instrument (Protein Solutions), the result showed a monomodal profiles centred at 4.4 nm radiuses corresponding to a molecular weight of 109 kDa.
Figure S3. Size exclusion gel filtration chromatography of the RraA from E. coli using a Superdex 200 column, the EcRraA protein was eluted as a single peak at volume 75.45 ml corresponding to a molecular weight of 87.7 kDa.
Figure S4. E coli RraA (PDB code 1Q5X) hexamer assembly.monomer a (cyan), b (salmon) and c (orange)assemble into one trimer which interaction with the other composing of d (green), e (blue) and f (marine) by hydrophilic force.
Figure S1.
Figure S2
Item / R (nm) / %Pd / MW-R (kDa) / %Int / %MassPeak 1 / 4.4 / 21.7 / 109 / 100.0 / 100.0
Figure S3
Figure S4