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- PDB-7kf5: Cryo-electron microscopy structure of the heavy metal efflux pump... -

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Basic information

Entry
Database: PDB / ID: 7kf5
TitleCryo-electron microscopy structure of the heavy metal efflux pump CusA in the symmetric closed state
ComponentsCation efflux system protein CusA
KeywordsMEMBRANE PROTEIN / TRANSPORT PROTEIN / efflux / pump / heavy metal. copper / silver / closed / open / transport
Function / homology
Function and homology information


silver ion transport / silver ion transmembrane transporter activity / plasma membrane copper ion transport / copper ion transmembrane transport / response to silver ion / silver ion transmembrane transport / copper ion export / copper ion transmembrane transporter activity / detoxification of copper ion / response to copper ion ...silver ion transport / silver ion transmembrane transporter activity / plasma membrane copper ion transport / copper ion transmembrane transport / response to silver ion / silver ion transmembrane transport / copper ion export / copper ion transmembrane transporter activity / detoxification of copper ion / response to copper ion / xenobiotic transmembrane transporter activity / intracellular copper ion homeostasis / response to toxic substance / copper ion binding / membrane / plasma membrane
Similarity search - Function
Cation efflux system CzcA/CusA/SilA/NccA/HelA/CnrA / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Cation efflux system protein CusA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMoseng, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI145069 United States
CitationJournal: mBio / Year: 2021
Title: Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export.
Authors: Mitchell A Moseng / Meinan Lyu / Tanadet Pipatpolkai / Przemyslaw Glaza / Corey C Emerson / Phoebe L Stewart / Phillip J Stansfeld / Edward W Yu /
Abstract: Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The CusA membrane protein, which ...Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer. The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.
History
DepositionOct 13, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Cation efflux system protein CusA
B: Cation efflux system protein CusA
C: Cation efflux system protein CusA


Theoretical massNumber of molelcules
Total (without water)347,5023
Polymers347,5023
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15860 Å2
ΔGint-72 kcal/mol
Surface area121020 Å2

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Components

#1: Protein Cation efflux system protein CusA


Mass: 115833.945 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cusA, ybdE, b0575, JW0564 / Production host: Escherichia coli (E. coli) / References: UniProt: P38054

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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: CusA in symmetric closed extrusion form / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.5 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13304 / Symmetry type: POINT

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