![]() Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy. doi: 10.7554/eLife.00594.īartesaghi A, Matthies D, Banerjee S, Merk A, Subramaniam S. Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K(+) channel. doi: 10.1126/science.aar4510.īanerjee A, Lee A, Campbell E, Mackinnon R. ![]() Structure of the human TRPM4 ion channel in a lipid nanodisc. doi: 10.1085/jgp.201411223.Īutzen HE, Myasnikov AG, Campbell MG, Asarnow D, Julius D, Cheng Y. K⁺ channel gating: C-type inactivation is enhanced by calcium or lanthanum outside. Acta Crystallographica Section D Biological Crystallography. PHENIX: a comprehensive Python-based system for macromolecular structure solution. The blue densities are shown from the same maps in gray, just at higher contour level and restricted to the central region of the pore.Īdams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH. From left to right, ~3.6 Å map of the entire complex obtained from 67,300 particles, ~3.8 Å map of the entire complex obtained from 75,633 particles, ~3.9 Å map of the entire complex obtained from 63,826 particles, ~3.9 Å map of the entire complex obtained from 118,556 particles, and ~4.6 Å map with local higher resolution in this central region of the transmembrane domain after signal subtraction and focused refinement of 34,654 particles (right). ![]() ( D) A variety of maps obtained during processing. ( C) Same view of the selectivity filter of the Kv chimera solved by X-ray crystallography (PDB entry 2r9r). ( B) Cryo-EM structure of the selectivity filter from the ~3.3 Å map of the entire complex of 47,482 particles. ( A) Transmembrane view of the selectivity filter of the Kv chimera solved by cryo-EM in lipid nanodiscs with densities in the pore from the ~4.0 Å map with local higher resolution in this central region of 65,745 signal subtracted particles including only the transmembrane region. For the TM domain, an overall 4 Å map was generated from 65,745 particles. A 3 Å map of the cytosolic domain was finally generated from 57,384 particles. Additional maps of the cytosolic domain and the transmembrane domain were obtained by a forced 3D classification of 94,131 particles into a single class, followed by signal subtraction and additional rounds of classifications and refinements. The particles yielding the map containing α and β subunits were used for 3D refinement to obtain the 3.3 Å map of the entire complex. ![]() To obtain the map of the entire complex, a round of 3D classification into 2 classes was carried out that divided the data into particles with both α and β subunits, and particles that seem to contain only α subunits. An additional round of 2D classification resulted in a selection of 94,131 particles. 118,556 particles indicating the presence of a nanodisc were left after multiple rounds of 2D and 3D classification, which were then re-extracted with a binning factor of 2 only using frames 2–20. Our findings show that large differences in structure between detergent and lipid bilayer environments are unlikely, and enable us to propose possible structural mechanisms for C-type inactivation.Ĭ-type inactivation Kv channel cryo-EM structure electron microscopy lipid nanodisc molecular biophysics neuroscience rat structural biology.Ģ81,021 particles were extracted from 2,062 micrographs using a binning factor of 4. ![]() At a resolution of ~3 Å for the cytosolic domain and ~4 Å for the transmembrane domain, the structure determined in nanodiscs is similar to the previously determined X-ray structure. Here we report the structure of the Kv1.2-2.1 paddle chimera channel reconstituted into lipid nanodiscs using single-particle cryo-electron microscopy. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. Voltage-activated potassium (Kv) channels open to conduct K + ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. ![]()
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