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Electron Microscopy of Ion Channels

English title Electron Microscopy of Ion Channels
Applicant Stahlberg Henning
Number 127545
Funding scheme Project funding (Div. I-III)
Research institution C-CINA Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Biophysics
Start/End 01.02.2010 - 31.01.2013
Approved amount 468'000.00
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Keywords (7)

CitS; MloK1; ClC-ec1; Ion Channels; Membrane Proteins; Electron Crystallography; 2D crystals

Lay Summary (English)

Lead
Membrane proteins are central to health and disease, and represent the major focus of intensive research efforts. We will analyze the structure of two ion channel membrane proteins in the membrane embedded state by electron microscopy.
Lay summary

The structure determination of human membrane proteins faces significant hurdles at several levels: It is difficult to obtain sufficient amounts of human membrane proteins for structure determination, and large well-ordered crystals are rarely obtained. The study of bacterial proteins as a replacement for human ones is easier, and the structures of such bacterial proteins can nevertheless teach us how the human proteins function. For the two here mentioned proteins we have obtained 2D crystals, where the membrane proteins are placed into artificial biological lipid membranes crystallized in these membranes. Such crystals can then be studied with the help of electron microscopes, and the 3D structure of the proteins is then determined by computer image processing.Aims:Here we will study the structure of two bacterial membrane proteins, one being an ion channel for chloride ions that on the same time anti-transports protons across the membranes, and the other being an ion channel for potassium ions that has gating functions. Both proteins can be produced in larger amounts and we have obtained well-ordered 2D crystals for both. We will study the 3D structure of these proteins under different conditions, as for example at different pH values or in dependence of the presence of certain ligands. The channels are then supposed to open or close under these conditions, so that we will also study the structural changes during channel gating.Significance:The studied bacterial proteins are representatives for important human membrane proteins that play key roles for muscle and nerve function, and are involved in osteoporosis, diabetes insipidus, and several other diseases. A better structural and functional understanding of these proteins may lead to the development of drugs to help patients that suffer from these medical conditions.

Direct link to Lay Summary Last update: 05.05.2013

Responsible applicant and co-applicants

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Publications

Publication
Projection Structure of the Secondary Citrate/Sodium Symporter CitS at 6 Å Resolution by Electron Crystallography.
Kebbel Fabian, Kurz Mareike, Grütter Markus G, Stahlberg Henning (2012), Projection Structure of the Secondary Citrate/Sodium Symporter CitS at 6 Å Resolution by Electron Crystallography., in Journal of molecular biology, 418(1-2), 117-26.
3D reconstruction from 2D crystal image and diffraction data.
Schenk Andreas D, Castaño-Díez Daniel, Gipson Bryant, Arheit Marcel, Zeng Xiangyan, Stahlberg Henning (2010), 3D reconstruction from 2D crystal image and diffraction data., in Methods in enzymology, 482, 101-29.
Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection.
Abeyrathne Priyanka D, Chami Mohamed, Pantelic Radosav S, Goldie Kenneth N, Stahlberg Henning (2010), Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection., in Methods in enzymology, 481, 25-43.
Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments.
Castaño-Díez Daniel, Kudryashev Mikhail, Arheit Marcel, Stahlberg Henning, Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments., in Journal of structural biology.

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Associated projects

Number Title Start Funding scheme
146929 Electron Microscopy of Membrane Proteins 01.04.2013 Project funding (Div. I-III)

Abstract

Membrane proteins are central to health and disease, and represent the major focus of intensive research efforts. Structure determination of membrane proteins faces hurdles, mostly in expression, purification, and 3D crystallization. Electron crystallography represents a valuable alternative for the structure determination of membrane proteins.We propose to structurally study three regulated ion channel membrane proteins by electron crystallography of 2D membrane crystals: The E. coli chloride channel ClC-ec1, now identified as chloride-proton antiporter, the M. loti cyclic nucleotide gated potassium channel MloK1, and the Ca2+ regulated potassium channel MthK from Methanobacterium autotrophicum. A crystallography structure of ClC-ec1 exists, but the available data do not allow establishing a conclusive model about the functioning and pH-dependent inhibition of the antiporter. For MloK1 only a structure of the cyclic nucleotide binding domain (CNBD) and the transmembrane part alone is available, but no data exist for the full-length molecule that demonstrate the action mechanism of cAMP binding to the CNBDs and their interaction with the putative voltage sensors. For MthK the mechanism of Ca2+ binding to the tetrameric or octameric RCK domains on channel opening/closing has not directly been demonstrated.We have obtained excellently ordered 2D crystals of ClC-ec1, with which we want to determine the membrane-embedded 3D structure at neutral pH by electron crystallography. Using electron diffraction and molecular replacement, we will then determine the structure at acidic pH. These data should allow determining the conformational changes associated with pH-dependent activation. We also have 2D crystals of MloK1. We can grow these crystals now also in the presence and absence of cAMP, which then show structurally different projection maps. We will elaborate the membrane-embedded 3D structure of this gated potassium channel, and study the mechanism of its regulation through cAMP, and the channel opening/closing effect on the orientation of the putative voltage sensor domains.We also have reconstituted MthK in lipid membranes, which always are stacked together. We will use electron tomography and single particle sub-volume averaging to study the intermediate-resolution structure of MthK in function of Ca2+ binding, and characterize the structural modifications for the channel dimensions.
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