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

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Basic information

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 / homology
Function and homology information


monoatomic ion transport / transmembrane transport / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Mechanosensitive ion channel MscS, conserved site / Uncharacterized protein family UPF0003 signature. / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel MscS / Mechanosensitive ion channel, beta-domain / Mechanosensitive ion channel MscS, beta-domain superfamily / LSM domain superfamily
Similarity search - Domain/homology
Low conductance mechanosensitive channel YnaI
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhang, Y. / Yu, J.
Funding support China, 5items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2016YFA0501100 China
Ministry of Science and Technology (China)2017YFA0504600 China
National Fund for Distinguished Young Scholar31625008 China
National Natural Science Foundation of China21532004 China
National Natural Science Foundation of China31570733 China
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.
History
DepositionAug 4, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 20, 2019Provider: repository / Type: Initial release

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Structure visualization

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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
Polymers277,3497
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area41000 Å2
ΔGint-294 kcal/mol
Surface area72190 Å2

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Components

#1: Protein
Low conductance mechanosensitive channel YnaI


Mass: 39621.215 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
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: all / 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 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / 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
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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