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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Bai, Jun-Ping; Moeini-Naghani, Iman; Zhong, Sheng et al. (2017) Current carried by the Slc26 family member prestin does not flow through the transporter pathway. Sci Rep 7:46619
Sigworth, Fred J (2016) Principles of cryo-EM single-particle image processing. Microscopy (Oxf) 65:57-67
Jensen, Katrine Hommelhoff; Sigworth, Fred J; Brandt, Sami Sebastian (2016) Removal of Vesicle Structures From Transmission Electron Microscope Images. IEEE Trans Image Process 25:540-52
Jensen, Katrine Hommelhoff; Brandt, Sami Sebastian; Shigematsu, Hideki et al. (2016) Statistical modeling and removal of lipid membrane projections for cryo-EM structure determination of reconstituted membrane proteins. J Struct Biol 194:49-60
Dvornek, Nicha C; Sigworth, Fred J; Tagare, Hemant D (2015) SubspaceEM: A fast maximum-a-posteriori algorithm for cryo-EM single particle reconstruction. J Struct Biol 190:200-14
Singh, Satinder K; Sigworth, Fred J (2015) Cryo-EM: Spinning the Micelles Away. Structure 23:1561
Tagare, Hemant D; Kucukelbir, Alp; Sigworth, Fred J et al. (2015) Directly reconstructing principal components of heterogeneous particles from cryo-EM images. J Struct Biol 191:245-62
Kucukelbir, Alp; Sigworth, Fred J; Tagare, Hemant D (2014) Quantifying the local resolution of cryo-EM density maps. Nat Methods 11:63-5
Liu, Yunhui; Sigworth, Fred J (2014) Automatic cryo-EM particle selection for membrane proteins in spherical liposomes. J Struct Biol 185:295-302
Shigematsu, H; Sigworth, F J (2013) Noise models and cryo-EM drift correction with a direct-electron camera. Ultramicroscopy 131:61-9

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