Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals.
The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis.
Hu
Guo-Bin
GB
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
Rice
William J
WJ
Dröse
Stefan
S
Altendorf
Karlheinz
K
Stokes
David L
DL
D000251:Adenosine Triphosphatases / Q000648:ultrastructure*; D027682:Cation Transport Proteins / Q000648:ultrastructure*; D003461:Crystallography; D004926:Escherichia coli / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000648:ultrastructure*; D000254:Sodium-Potassium-Exchanging ATPase / Q000648:ultrastructure*; D014054:Tomography
D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D000254:Sodium-Potassium-Exchanging ATPase