TssA from Aeromonas hydrophila: expression, purification and crystallographic studies

Abstract

Crystals of different domain constructs of the TssA subunit from Aeromonas hydrophila have been crystallized in forms suitable for X-ray analysis.

Structural insights into the function of type VI secretion system TssA subunits

“…The structure of an Ah TssA2 A Nt2 domain construct (D231-L374) was solved to 1.76 Å with four subunits (chains A–D) in the asymmetric unit (Supplementary Table 6 ) arranged as two independent dimers, consistent with previous SEC analysis 33 , that showed that Ah TssA2 A Nt2 exists as dimers in solution. Each monomer folds into a cluster of seven α-helices (α1–α7), the arrangement of which is very similar to that of the EAEC TssA2 B Nt2 domain (RMSD = 2.0 Å) as determined by structural alignment 27 , 34 .…”

“…Expression and purification of His 6 .TssA2 A (1–478), His 6 .TssA2 A Nt2 (223–387), His 6 .TssA2 A Nt2-CTD (223–478) and TssA2 A CTD (381–478) was carried out as previously described 33 . Both MBP.TssA1 B CTD (303–373) and His 6 .TssA1 B Nt1 (1–255) were overexpressed and purified, with TssA1 B CTD (303–373) being separated from the MBP solubility tag by Factor Xa cleavage 51 .…”

“…To generate a derivative for crystals produced from purified His 6 .TssA2 A Nt2 (223–387) 33 , flakes of ethyl mercury phosphate powder were added to a drop containing crystals for 5 min, before subsequent cryoprotection in 0.04 M potassium phosphate monobasic, 16% (w/v) PEG 8000, 30% (v/v) glycerol. Hg SAD data were collected at 1.0088 Å wavelength on beamline I03 at the Diamond Light Source, and data were processed in xia2-DIALS 54 – 58 to 2.16 Å in spacegroup P2 1 (Supplementary Table 6 ).…”

“…Native data on crystals of His 6 .TssA2 A Nt2-CTD (223–478) 33 were collected on beamline I03 at the Diamond Light Source. Data were processed in xia2-DIALS 54 – 56 , 58 to 3.13 Å, in spacegroup P2 1 (Supplementary Table 7 ).…”