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- PDB-7n4t: Low conductance mechanosensitive channel YnaI -

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Basic information

Entry
Database: PDB / ID: 7n4t
TitleLow conductance mechanosensitive channel YnaI
ComponentsLow conductance mechanosensitive channel YnaI
KeywordsTRANSPORT PROTEIN / Low conductance mechanosensitive channel YnaI / NCMN / ion channel / membrane protein
Function / homology
Function and homology information


mechanosensitive monoatomic ion channel activity / cellular response to osmotic stress / identical protein binding / plasma membrane
Similarity search - Function
Mechanosensitive ion channel protein YnaI-like / : / : / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel, transmembrane helices 2/3 / 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 protein YnaI-like / : / : / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel, transmembrane helices 2/3 / 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
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Low conductance mechanosensitive channel YnaI
Similarity search - Component
Biological speciesEscherichia coli 'BL21-GoldpLysS AG'
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / cryo EM / Resolution: 2.4 Å
AuthorsCatalano, C. / Ben-Hail, D. / Qiu, W. / des Georges, A. / Guo, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM132329 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R35GM133598 United States
CitationJournal: Membranes (Basel) / Year: 2021
Title: Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities.
Authors: Claudio Catalano / Danya Ben-Hail / Weihua Qiu / Paul Blount / Amedee des Georges / Youzhong Guo /
Abstract: Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution ...Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.
History
DepositionJun 4, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,75514
Polymers271,5467
Non-polymers5,2087
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area50010 Å2
ΔGint-408 kcal/mol
Surface area67880 Å2

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Components

#1: Protein
Low conductance mechanosensitive channel YnaI


Mass: 38792.344 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: K12 / Gene: ynaI, b1330, JW1323
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0AEB5
#2: Chemical
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 744.034 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C41H78NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: DOPE, phospholipid*YM
Has ligand of interestY

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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: Low conductance mechanosensitive channel YnaI / Type: CELL / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: YES
EM stainingType: NEGATIVE / Material: 2 % uranil acetate
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 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 recordingAverage exposure time: 8 sec. / Electron dose: 56.18 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2155
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2Leginonimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 330000
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 5Y4O

5y4o

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313160
ELECTRON MICROSCOPYf_angle_d0.50817829
ELECTRON MICROSCOPYf_dihedral_angle_d7.7481876
ELECTRON MICROSCOPYf_chiral_restr0.0422037
ELECTRON MICROSCOPYf_plane_restr0.0042226

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