Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy

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Abstract

A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography1 in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods1 in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM))2. Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel3 (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7–2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.

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Figure 1: BK channel structure and specific potassium permeability.
Figure 2: Cryo-EM specimen and image processing.
Figure 3: Structure of the transmembrane region.
Figure 4: Structure of the gating ring.

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Data deposits

The density maps are deposited in the Electron Microscopy Data Bank under accession numbers EMD-5114 and EMD-5121.

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Acknowledgements

We thank N. Grigorieff and C. Xu for use of the Tecnai F30 microscope and help with data collection. We also thank S. Bian for sharing his BK purification methods, and Y. Yan for cell culture. Image processing and reconstruction made use of the Yale Biomedical High-Performance Computing Center.

Author Contributions L.W. and F.J.S. designed the experiments; L.W. purified and reconstituted the protein, made and imaged the cryo-EM specimens, and performed image processing and reconstruction. L.W. wrote custom software, with some algorithms contributed by F.J.S. L.W. and F.J.S. co-wrote the paper.

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Correspondence to Fred J. Sigworth.

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Wang, L., Sigworth, F. Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Nature 461, 292–295 (2009) doi:10.1038/nature08291

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