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- PDB-7a46: small conductance mechanosensitive channel YbiO -

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Basic information

Entry
Database: PDB / ID: 7a46
Titlesmall conductance mechanosensitive channel YbiO
ComponentsPutative transport protein
KeywordsMEMBRANE PROTEIN / YbiO / mechanosensitive channel / MscS-like channel / E. coli
Function / homology
Function and homology information


ion transport / transmembrane transport / integral component of membrane / plasma membrane
Mechanosensitive ion channel MscS / LSM domain superfamily / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS domain superfamily
Putative transport protein
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsFlegler, V.J. / Rasmussen, A. / Rao, S. / Wu, N. / Zenobi, R. / Sansom, M.S.P. / Hedrich, R. / Rasmussen, T. / Boettcher, B.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)Bo1150/15-1 Germany
German Research Foundation (DFG)INST 93/903-1 FUGG Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: The MscS-like channel YnaI has a gating mechanism based on flexible pore helices.
Authors: Vanessa Judith Flegler / Akiko Rasmussen / Shanlin Rao / Na Wu / Renato Zenobi / Mark S P Sansom / Rainer Hedrich / Tim Rasmussen / Bettina Böttcher /
Abstract: The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. ...The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 19, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Putative transport protein
B: Putative transport protein
C: Putative transport protein
D: Putative transport protein
E: Putative transport protein
F: Putative transport protein
G: Putative transport protein


Theoretical massNumber of molelcules
Total (without water)590,8657
Polymers590,8657
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28580 Å2
ΔGint-191 kcal/mol
Surface area70970 Å2
MethodPISA

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Components

#1: Protein
Putative transport protein /


Mass: 84409.242 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ybiO, BN17_06071 / Production host: Escherichia coli (E. coli) / References: UniProt: J7R2E5

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: mechanosensitive channel YbiO / Type: COMPLEX / Details: YbiO reconstituted in amphipol A8-35 / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.566 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 65 sec. / Electron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4145
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7Coot0.8.9model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 419249 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 159 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 6RLD
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00611669
ELECTRON MICROSCOPYf_angle_d0.55715883
ELECTRON MICROSCOPYf_dihedral_angle_d20.4794039
ELECTRON MICROSCOPYf_chiral_restr0.0411967
ELECTRON MICROSCOPYf_plane_restr0.0022030

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