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- PDB-5y4o: Cryo-EM structure of MscS channel, YnaI -

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Entry
Database: PDB / ID: 5y4o
TitleCryo-EM structure of MscS channel, YnaI
ComponentsLow conductance mechanosensitive channel YnaI
KeywordsMEMBRANE PROTEIN / cryo-EM / MscS / Na/K selective channel
Function / homologyMechanosensitive ion channel MscS / Mechanosensitive ion channel MscS, conserved site / LSM domain superfamily / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS domain superfamily / Mechanosensitive ion channel / Uncharacterized protein family UPF0003 signature. / ion transport / transmembrane transport ...Mechanosensitive ion channel MscS / Mechanosensitive ion channel MscS, conserved site / LSM domain superfamily / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS domain superfamily / Mechanosensitive ion channel / Uncharacterized protein family UPF0003 signature. / ion transport / transmembrane transport / integral component of membrane / plasma membrane / Low conductance mechanosensitive channel YnaI
Function and homology information
Specimen sourceEscherichia coli O157:H7 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhang, Y. / Yu, J.
Funding supportChina , 5 items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2016YFA0501100China
Ministry of Science and Technology (China)2017YFA0504600China
National Fund for Distinguished Young Scholar31625008China
National Natural Science Foundation of China21532004China
National Natural Science Foundation of China31570733China
CitationJournal: Protein Cell / Year: 2018
Title: A binding-block ion selective mechanism revealed by a Na/K selective channel.
Authors: Jie Yu / Bing Zhang / Yixiao Zhang / Cong-Qiao Xu / Wei Zhuo / Jingpeng Ge / Jun Li / Ning Gao / Yang Li / Maojun Yang /
Abstract: Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of ...Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na/K cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI facilitate Na/K pass through, which was defined as binding-block mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 4, 2017 / Release: Mar 20, 2019

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Assembly

Deposited unit
A: Low conductance mechanosensitive channel YnaI
B: Low conductance mechanosensitive channel YnaI
C: Low conductance mechanosensitive channel YnaI
D: Low conductance mechanosensitive channel YnaI
E: Low conductance mechanosensitive channel YnaI
F: Low conductance mechanosensitive channel YnaI
G: Low conductance mechanosensitive channel YnaI


Theoretical massNumber of molelcules
Total (without water)277,3497
Polyers277,3497
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)41000
ΔGint (kcal/M)-294
Surface area (Å2)72190

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Components

#1: Protein/peptide
Low conductance mechanosensitive channel YnaI


Mass: 39621.215 Da / Num. of mol.: 7 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: ynaI, Z2437, ECs1912
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0AEB6

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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: YnaI / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli O157:H7 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 6.5
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277 kelvins

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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
Image recordingElectron dose: 37 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42000 / Symmetry type: POINT

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