TssA from Burkholderia cenocepacia: expression, purification, crystallization and crystallographic analysis

Abstract

Analysis of the noncrystallographic symmetry of crystals of the C-terminal domain of Burkholderia cenocepacia TssA indicates a quaternary structure of 32 subunits in D 16 symmetry.

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

“…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 . MBP.TssA1 B CTD (294–373) was produced and purified in a similar manner as MBP.TssA1 B CTD (303–373).…”

“…For crystallisation, MBP.His 6 .TssA1 B CTD H12–H14 (amino acids 303–358) was purified by amylose affinity chromatography, and eluted with 10 mM maltose. His 6 .TssA1 B CTD H12–H14 was proteolytically removed from the MBP tag by treatment with Factor Xa, followed by purification on a nickel column with imidazole gradient elution and SEC on a Superdex 200 column prior to concentration and final buffer exchange 51 .…”

“…Native data on crystals of purified His 6 .TssA1 B Nt1 (1–255) 51 were collected on beamline I02 at the Diamond Light Source, and 150° of data were processed in xia2–3d 54 – 57 to 1.8 Å in spacegroup P2 1 2 1 2 (Supplementary Table 2 ). Crystals from the same conditions were subjected to sublimation of elemental iodine crystals overnight.…”

“…Native data on crystals of purified TssA1 B CTD (303–373) 51 and the selenomethionine derivative were collected on beamline I24 at the Diamond Light Source. Data were processed in xia2–3da 54 – 56 , 63 , 64 (Supplementary Table 3 ) to 3.08 Å, in spacegroup I222.…”

“…Native data were collected on beamline I04–1 at the Diamond Light Source, and processed in xia2 3daii 54 – 57 , 63 , 64 to 1.78 Å in spacegroup I222 (Supplementary Table 5 ). Selenomethionine-incorporated MBP.TssA1 B CTD (303–373) was overexpressed and purified using a similar strategy to that described previously 51 . Crystals were produced in condition 0.1 M citric acid (pH 5.0), 20% (w/v) PEG 6000, and were cryoprotected in 0.1 M citric acid (pH 4.0), 22% (w/v) PEG 6000, 25% (v/v) glycerol.…”