A long-pursued goal of structural biology has been to determine the structure of membrane proteins in their membrane environment, ideally in their various functional states as defined for example by ion gradients and membrane potentials. In the proposed work we will apply a new, general method for single-particle cryo-EM structure determination of membrane proteins reconstituted into liposomes. The method is called random spherically-constrained (RSC) single-particle reconstruction and involves the acquisition and processing of images of lipid vesicles containing the protein of interest and having the desired transmembrane gradients imposed.
The aims of the project are first to optimize the specimen, imaging and processing strategies for this process, and then apply it to structure determination of Kv and BK potassium channels in their various states. The goal is to observe the conformational changes that underlie the channel gating processes, in particular the rearrangements of voltage-sensor domains of both channel types and the calcium-sensing gating ring of the BK channel.

Public Health Relevance

Ion channels are molecular machines responsible for the electrical activity, normal and aberrant, of cells in the brain, heart, and many other tissues. The gate the flow of ions across membranes in response to voltage changes and intracellular-messenger molecules. Mapping the three-dimensional structure of these machines in their various states will clarify the mechanisms underlying their responses.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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Silberberg, Shai D
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Yale University
Schools of Medicine
New Haven
United States
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Liu, Yunhui; Sigworth, Fred J (2014) Automatic cryo-EM particle selection for membrane proteins in spherical liposomes. J Struct Biol 185:295-302
Kucukelbir, Alp; Sigworth, Fred J; Tagare, Hemant D (2014) Quantifying the local resolution of cryo-EM density maps. Nat Methods 11:63-5
Miranda, Pablo; Contreras, Jorge E; Plested, Andrew J R et al. (2013) State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels. Proc Natl Acad Sci U S A 110:5217-22
Shigematsu, H; Sigworth, F J (2013) Noise models and cryo-EM drift correction with a direct-electron camera. Ultramicroscopy 131:61-9
Wang, Liguo (2012) Measurements and implications of the membrane dipole potential. Annu Rev Biochem 81:615-35
Bian, Shumin; Bai, Jun-Ping; Chapin, Hannah et al. (2011) Interactions between ?-catenin and the HSlo potassium channel regulates HSlo surface expression. PLoS One 6:e28264
Syed, Sheyum; Mullner, Fiona E; Selvin, Paul R et al. (2010) Improved hidden Markov models for molecular motors, part 2: extensions and application to experimental data. Biophys J 99:3696-703
Mullner, Fiona E; Syed, Sheyum; Selvin, Paul R et al. (2010) Improved hidden Markov models for molecular motors, part 1: basic theory. Biophys J 99:3684-95
Wang, Liguo; Sigworth, Fred J (2009) Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Nature 461:292-5
Wang, Liguo; Ounjai, Puey; Sigworth, Fred J (2008) Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection. J Struct Biol 164:190-8

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