Investigator
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Misha Kudryashev
Group Leader
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Max Planck Institute of Biophysics &
Buchmann Institute for Molecular Life Sciences (BMLS)
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 3
60438 Frankfurt am Main, Germany
Phone +49 (0)69 6303-1700
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Native structure of ion channels in membranes under voltage
Ion channels are critical for transmission the signals between cells or cells and extracellular space. This is especially important in the neurons where communication in the synapse leads to quick synaptic activation and long-term memory generation. Knowing the structure of the ion channels is instrumental to being able to understand their function and ultimately design pharmacological compounds. We use modern methods for cyro electron microscopy and tomography [1] to determine the structure of ion channels in their native state – in membranes and under concentration gradients applied across the membranes. For this we express, purify and reconstitute the proteins into the closed lipid vesicles (Figure 1). Physiological gradients may be imposed by replacing the buffer inside and outside of the vesicles.
Figure 1. Structure of the mouse serotonin receptor in lipid vesicles [1].
A, B: Reconstituted receptors are imaged using EM (A) and low dose cryo electron tomography (B). Thousands of protein copies from B are mutually aligned and summed up resulting in a reconstruction at 12 Å resolution (C) where it is possible to fit the known high resolution structure (D). Scale bars: 50 nm in A, B and 5 nm in C.
For the structural analysis we use a multi-scale approach combining high resolution structural analysis with cryo electron tomography and subtomogram averaging [1]. While the recently revolutionized methods of single particle cryo-EM result in atomic resolution (3-4 Å) structures for a large number of proteins and protein complexes [2], it can be applied on the isolated proteins only. Subtomogram averaging may give information about more complex topologies but the resolution is limited to a roughly 10 Å. The major effort is to bring the resolution of subtomogram averaging higher by developing better data processing routines ourselves and in collaborations [3-5]. EM maps at 7 Å resolution in combination with knowing the atomic structure and computational methods give unprecedented insights into the gating of ion channels.
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Publications
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- Kudryashev M, Castaño-Díez D, Deluz C, Hassaine G, Grasso L, Graf-Meyer A, Vogel H, Stahlberg H (2016) The Structure of the Mouse Serotonin 5-HT 3 Receptor in Lipid Vesicles. Structure 24, 165-170.
- Kudryashev M, Wang RY, Brackmann M, Scherer S, Maier T, Baker D, DiMaio F, Stahlberg H, Egelman EH, Basler M (2015) Structure of the type VI secretion system contractile sheath. Cell 160, 952-62.
- Castaño-Díez D, Kudryashev M, Arheit M, Stahlberg H (2012) Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J Struct Biol 178, 139-51.
- Castaño-Díez D, Kudryashev M, Stahlberg H (2016) Dynamo Catalogue: Geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms. J Struct Biol, doi: 10.1016/j.jsb.2016.06.005
- Wang RYR, Kudryashev M, Li X, Egelman H, Basler M, Cheng Y, Baker D, DiMaio F (2015) De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nature Methods 12, 335-8.