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- PDB-7kf8: Cryo-electron microscopy structure of the heavy metal efflux pump... -

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Basic information

Entry
Database: PDB / ID: 7kf8
TitleCryo-electron microscopy structure of the heavy metal efflux pump CusA in a heterogeneous 2 open and 1 closed protomer conformation
ComponentsCation efflux system protein CusA
KeywordsMEMBRANE PROTEIN / TRANSPORT PROTEIN / efflux / pump / heavy metal. copper / silver / closed / open / transporter
Function / homology
Function and homology information


silver ion transmembrane transporter activity / silver ion transport / plasma membrane copper ion transport / response to silver ion / copper ion export / copper ion transmembrane transporter activity / detoxification of copper ion / xenobiotic transmembrane transporter activity / response to copper ion / cellular copper ion homeostasis ...silver ion transmembrane transporter activity / silver ion transport / plasma membrane copper ion transport / response to silver ion / copper ion export / copper ion transmembrane transporter activity / detoxification of copper ion / xenobiotic transmembrane transporter activity / response to copper ion / cellular copper ion homeostasis / copper ion binding / integral component of membrane / plasma membrane
AcrB/AcrD/AcrF family / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / Cation efflux system CzcA/CusA/SilA/NccA/HelA/CnrA / Acriflavin resistance protein
Cation efflux system protein CusA
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 13, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)347,6295
Polymers347,5023
Non-polymers1272
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17880 Å2
ΔGint-125 kcal/mol
Surface area123150 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
#2: Chemical ChemComp-CU1 / COPPER (I) ION / Copper


Mass: 63.546 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
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: CusA with heterogeneous protomer conformations / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding 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 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43395 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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