Journal: Structure / Year: 2016 Title: The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles. Authors: Mikhail Kudryashev / Daniel Castaño-Díez / Cédric Deluz / Gherici Hassaine / Luigino Grasso / Alexandra Graf-Meyer / Horst Vogel / Henning Stahlberg / Abstract: The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid ...The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.
History
Deposition
Jul 24, 2015
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Header (metadata) release
Aug 19, 2015
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Map release
Jan 13, 2016
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Update
Jan 27, 2016
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Current status
Jan 27, 2016
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
EMPIAR-10046 (Title: Cryo electron tomography of mouse 5-HT3 receptors in lipid vesicles Data size: 158.8 Data #1: Tilt series of the 5-HT3 receptors reconstituted to lipid vesicles [class averages])
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: LEICA EM GP / Method: Blot for 2 second before plunding
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Electron microscopy
Microscope
FEI TITAN KRIOS
Alignment procedure
Legacy - Astigmatism: corrected at high magnification
Details
tomograms acquired without the energy filtration
Date
Dec 1, 2013
Image recording
Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 46 / Average electron dose: 41 e/Å2 / Details: 46 tomograms were selected for processing
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Details
Particles were automatically picked from the surface of the vesicles in CTF-corrected cryo-electron tomograms. After a rough alignment deep classification into 20 classes was performed including 4 neighbouring pentamers in the alignment mask. Finally the central pentamers from each class were cropped, aligned and averaged resulting in a final structure.
Final reconstruction
Applied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: OTHER / Software - Name: IMOD, Dynamo / Number subtomograms used: 16000
CTF correction
Details: ctfplotter
Final 3D classification
Number classes: 20
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