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- EMDB-22846: Cryo-electron microscopy structure of the heavy metal efflux pump... -

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Basic information

Entry
Database: EMDB / ID: EMD-22846
TitleCryo-electron microscopy structure of the heavy metal efflux pump CusA in a heterogeneous 2 open and 1 closed protomer conformation
Map data
Sample
  • Complex: CusA with heterogeneous protomer conformations
    • Protein or peptide: Cation efflux system protein CusA
  • Ligand: COPPER (I) ION
Keywordsefflux / pump / heavy metal. copper / silver / closed / open / transporter / MEMBRANE PROTEIN / TRANSPORT PROTEIN
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 transmembrane transporter activity / copper ion export / 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 transmembrane transporter activity / copper ion export / 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 / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / Acriflavin resistance protein / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Cation efflux system protein CusA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMoseng MA
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseApr 14, 2021-
Map releaseApr 14, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kf8
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22846.png

Downloads & links

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Map

FileDownload / File: emd_22846.map.gz / Format: CCP4 / Size: 12.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 143 pix.
= 154.44 Å		1.08 Å/pix.
x 147 pix.
= 158.76 Å		1.08 Å/pix.
x 156 pix.
= 168.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.6848532 - 1.3776424
Average (Standard dev.)0.000000000001341 (±0.109616995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin10593105
Dimensions147156143
Spacing143147156
CellA: 154.44 Å / B: 158.76001 Å / C: 168.48001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z143147156
origin x/y/z0.0000.0000.000
length x/y/z154.440158.760168.480
α/β/γ90.00090.00090.000
start NX/NY/NZ10510593
NX/NY/NZ143147156
MAP C/R/S321
start NC/NR/NS93105105
NC/NR/NS156147143
D min/max/mean-0.6851.3780.000

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Supplemental data

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Sample components

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Entire : CusA with heterogeneous protomer conformations

EntireName: CusA with heterogeneous protomer conformations
Components
  • Complex: CusA with heterogeneous protomer conformations
    • Protein or peptide: Cation efflux system protein CusA
  • Ligand: COPPER (I) ION

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Supramolecule #1: CusA with heterogeneous protomer conformations

SupramoleculeName: CusA with heterogeneous protomer conformations / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Cation efflux system protein CusA

MacromoleculeName: Cation efflux system protein CusA / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 115.833945 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHMI EWIIRRSVAN RFLVLMGALF LSIWGTWTII NTPVDALPDL SDVQVIIKTS YPGQAPQIVE NQVTYPLTTT MLSVPGAKT VRGFSQFGDS YVYVIFEDGT DPYWARSRVL EYLNQVQGKL PAGVSAELGP DATGVGWIYE YALVDRSGKH D LADLRSLQ ...String:
MGHHHHHHMI EWIIRRSVAN RFLVLMGALF LSIWGTWTII NTPVDALPDL SDVQVIIKTS YPGQAPQIVE NQVTYPLTTT MLSVPGAKT VRGFSQFGDS YVYVIFEDGT DPYWARSRVL EYLNQVQGKL PAGVSAELGP DATGVGWIYE YALVDRSGKH D LADLRSLQ DWFLKYELKT IPDVAEVASV GGVVKEYQVV IDPQRLAQYG ISLAEVKSAL DASNQEAGGS SIELAEAEYM VR ASGYLQT LDDFNHIVLK ASENGVPVYL RDVAKVQIGP EMRRGIAELN GEGEVAGGVV ILRSGKNARE VIAAVKDKLE TLK SSLPEG VEIVTTYDRS QLIDRAIDNL SGKLLEEFIV VAVVCALFLW HVRSALVAII SLPLGLCIAF IVMHFQGLNA NIMS LGGIA IAVGAMVDAA IVMIENAHKR LEEWQHQHPD ATLDNKTRWQ VITDASVEVG PALFISLLII TLSFIPIFTL EGQEG RLFG PLAFTKTYAM AGAALLAIVV IPILMGYWIR GKIPPESSNP LNRFLIRVYH PLLLKVLHWP KTTLLVAALS VLTVLW PLN KVGGEFLPQI NEGDLLYMPS TLPGISAAEA ASMLQKTDKL IMSVPEVARV FGKTGKAETA TDSAPLEMVE TTIQLKP QE QWRPGMTMDK IIEELDNTVR LPGLANLWVP PIRNRIDMLS TGIKSPIGIK VSGTVLADID AMAEQIEEVA RTVPGVAS A LAERLEGGRY INVEINREKA ARYGMTVADV QLFVTSAVGG AMVGETVEGI ARYPINLRYP QSWRDSPQAL RQLPILTPM KQQITLADVA DIKVSTGPSM LKTENARPTS WIYIDARDRD MVSVVHDLQK AIAEKVQLKP GTSVAFSGQF ELLERANHKL KLMVPMTLM IIFVLLYLAF RRVGEALLII SSVPFALVGG IWLLWWMGFH LSVATGTGFI ALAGVAAEFG VVMLMYLRHA I EAVPSLNN PQTFSEQKLD EALYHGAVLR VRPKAMTVAV IIAGLLPILW GTGAGSEVMS RIAAPMIGGM ITAPLLSLFI IP AAYKLMW LHRHRVRK

UniProtKB: Cation efflux system protein CusA

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Macromolecule #2: COPPER (I) ION

MacromoleculeName: COPPER (I) ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CU1
Molecular weightTheoretical: 63.546 Da
Chemical component information

ChemComp-CU1:
COPPER (I) ION

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.5 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 43395
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7kf8:
Cryo-electron microscopy structure of the heavy metal efflux pump CusA in a heterogeneous 2 open and 1 closed protomer conformation

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