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- EMDB-16123: Cryo-EM structure of the wild-type solitary ECF module in DDM mic... -

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Basic information

Entry
Database: EMDB / ID: EMD-16123
TitleCryo-EM structure of the wild-type solitary ECF module in DDM micelles in the ATPase open and nucleotide-free conformation
Map dataATPase open and nucleotide-free conformation of the wild-type solitary ECF module in MSP2N2 lipid nanodiscs at 4.3 A resolution sharpened at -159 A^2.
Sample
  • Complex: Wild-type solitary ECF module
    • Protein or peptide: Energy-coupling factor transporter ATP-binding protein EcfA1
    • Protein or peptide: Energy-coupling factor transporter ATP-binding protein EcfA2
    • Protein or peptide: Energy-coupling factor transporter transmembrane protein EcfT
KeywordsABC Transporter / ECF transporter complex / motor / membrane protein
Function / homology
Function and homology information


Translocases / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
ECF transporter transmembrane protein EcfT / Energy-coupling factor transporter ATP-binding protein EcfA2 / Energy-coupling factor transporter ATP-binding protein EcfA1 / ABC/ECF transporter, transmembrane component / Cobalt transport protein / Cobalt import ATP-binding protein cbiO. / ABC transporter, CbiO/EcfA subunit / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter ...ECF transporter transmembrane protein EcfT / Energy-coupling factor transporter ATP-binding protein EcfA2 / Energy-coupling factor transporter ATP-binding protein EcfA1 / ABC/ECF transporter, transmembrane component / Cobalt transport protein / Cobalt import ATP-binding protein cbiO. / ABC transporter, CbiO/EcfA subunit / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Energy-coupling factor transporter transmembrane protein EcfT / Energy-coupling factor transporter ATP-binding protein EcfA2 / Energy-coupling factor transporter ATP-binding protein EcfA1
Similarity search - Component
Biological speciesLactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsThangaratnarajah C / Rheinberger J / Paulino C / Slotboom DJ
Funding supportEuropean Union, Netherlands, 5 items
OrganizationGrant numberCountry
European Union (EU)847675European Union
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)40.018.016 Netherlands
Netherlands Organisation for Scientific Research (NWO)714.018.003 Netherlands
European Research Council (ERC)812867European Union
CitationJournal: Nat Commun / Year: 2023
Title: Expulsion mechanism of the substrate-translocating subunit in ECF transporters.
Authors: Chancievan Thangaratnarajah / Mark Nijland / Luís Borges-Araújo / Aike Jeucken / Jan Rheinberger / Siewert J Marrink / Paulo C T Souza / Cristina Paulino / Dirk J Slotboom /
Abstract: Energy-coupling factor (ECF)-type transporters mediate the uptake of micronutrients in many bacteria. They consist of a substrate-translocating subunit (S-component) and an ATP-hydrolysing motor (ECF ...Energy-coupling factor (ECF)-type transporters mediate the uptake of micronutrients in many bacteria. They consist of a substrate-translocating subunit (S-component) and an ATP-hydrolysing motor (ECF module) Previous data indicate that the S-component topples within the membrane to alternately expose the binding site to either side of the membrane. In many ECF transporters, the substrate-free S-component can be expelled from the ECF module. Here we study this enigmatic expulsion step by cryogenic electron microscopy and reveal that ATP induces a concave-to-convex shape change of two long helices in the motor, thereby destroying the S-component's docking site and allowing for its dissociation. We show that adaptation of the membrane morphology to the conformational state of the motor may favour expulsion of the substrate-free S-component when ATP is bound and docking of the substrate-loaded S-component after hydrolysis. Our work provides a picture of bilayer-assisted chemo-mechanical coupling in the transport cycle of ECF transporters.
History
DepositionNov 10, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16123.png

Downloads & links

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Map

FileDownload / File: emd_16123.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationATPase open and nucleotide-free conformation of the wild-type solitary ECF module in MSP2N2 lipid nanodiscs at 4.3 A resolution sharpened at -159 A^2.
Voxel sizeX=Y=Z: 1.022 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.1366136 - 1.876189
Average (Standard dev.)0.0033607874 (±0.050733354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 261.632 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16123_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map obtained with DeepEMhancer used for figures.

Fileemd_16123_additional_1.map
AnnotationSharpened map obtained with DeepEMhancer used for figures.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 used during refinement and FSC...

Fileemd_16123_half_map_1.map
AnnotationHalf map 1 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 used during refinement and FSC...

Fileemd_16123_half_map_2.map
AnnotationHalf map 2 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Wild-type solitary ECF module

EntireName: Wild-type solitary ECF module
Components
  • Complex: Wild-type solitary ECF module
    • Protein or peptide: Energy-coupling factor transporter ATP-binding protein EcfA1
    • Protein or peptide: Energy-coupling factor transporter ATP-binding protein EcfA2
    • Protein or peptide: Energy-coupling factor transporter transmembrane protein EcfT

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Supramolecule #1: Wild-type solitary ECF module

SupramoleculeName: Wild-type solitary ECF module / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)

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Macromolecule #1: Energy-coupling factor transporter ATP-binding protein EcfA1

MacromoleculeName: Energy-coupling factor transporter ATP-binding protein EcfA1
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: GSDNIISFDH VTFTYPDSPR PALSDLSFAI ERGSWTALIG HNGSGKSTVS KLINGLLAPD DLDKSSITVD GVKLGADTVW EVREKVGIVF QNPDNQFVGA TVSDDVAFGL ENRAVPRPEM LKIVAQAVAD VGMADYADSE PSNLSGGQKQ RVAIAGILAV KPQVIILDES ...String:
GSDNIISFDH VTFTYPDSPR PALSDLSFAI ERGSWTALIG HNGSGKSTVS KLINGLLAPD DLDKSSITVD GVKLGADTVW EVREKVGIVF QNPDNQFVGA TVSDDVAFGL ENRAVPRPEM LKIVAQAVAD VGMADYADSE PSNLSGGQKQ RVAIAGILAV KPQVIILDES TSMLDPEGKE QILDLVRKIK EDNNLTVISI THDLEEAAGA DQVLVLDDGQ LLDQGKPEEI FPKVEMLKRI GLDIPFVYRL KQLLKERGIV LPDEIDDDEK LVQSLWQLNS KM

UniProtKB: Energy-coupling factor transporter ATP-binding protein EcfA1

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Macromolecule #2: Energy-coupling factor transporter ATP-binding protein EcfA2

MacromoleculeName: Energy-coupling factor transporter ATP-binding protein EcfA2
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MAIKFENVSY VYSPGSPLEA IGLDQLNFSL EEGKFIALVG HTGSGKSTLM QHFNALLKPT SGKIEIAGYT ITPETGNKGL KDLRRKVSLA FQFSEAQLFE NTVLKDVEYG PRNFGFSEDE AREAALKWLK KVGLKDDLIE HSPFDLSGGQ MRRVALAGVL AYEPEIICLD ...String:
MAIKFENVSY VYSPGSPLEA IGLDQLNFSL EEGKFIALVG HTGSGKSTLM QHFNALLKPT SGKIEIAGYT ITPETGNKGL KDLRRKVSLA FQFSEAQLFE NTVLKDVEYG PRNFGFSEDE AREAALKWLK KVGLKDDLIE HSPFDLSGGQ MRRVALAGVL AYEPEIICLD EPAAGLDPMG RLEMMQLFKD YQAAGHTVIL VTHNMDDVAD YADDVLALEH GRLIKHASPK EVFKDSEWLQ KHHLAEPRSA RFAAKLEAAG LKLPGQPLTM PELADAIKQS LKGGEHE

UniProtKB: Energy-coupling factor transporter ATP-binding protein EcfA2

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Macromolecule #3: Energy-coupling factor transporter transmembrane protein EcfT

MacromoleculeName: Energy-coupling factor transporter transmembrane protein EcfT
type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MSKIIIGRYL PGTTFVYRVD PRAKLLTTFY FIIMIFLANN WVSYLVISIF GLAYVFATGL KARVFWDGVK PMIWMIVFTS LLQTFFMAGG KVYWHWWIFT LSSEGLINGL YVFIRFAMII LVSTVMTVTT KPLEIADAME WMLTPLKLFK VNVGMISLVI SIALRFVPTL ...String:
MSKIIIGRYL PGTTFVYRVD PRAKLLTTFY FIIMIFLANN WVSYLVISIF GLAYVFATGL KARVFWDGVK PMIWMIVFTS LLQTFFMAGG KVYWHWWIFT LSSEGLINGL YVFIRFAMII LVSTVMTVTT KPLEIADAME WMLTPLKLFK VNVGMISLVI SIALRFVPTL FDQTVKIMNA QRSRGADFND GGLVKRAKSV VPMLVPLFID SLEVALDLST AMESRGYKGS EGRTRYRILE WSKVDLIPVA YCLLLTILMI TTRKH

UniProtKB: Energy-coupling factor transporter transmembrane protein EcfT

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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

Concentration5.4 mg/mL
BufferpH: 8 / Details: 20 mM Tris, pH 8.0, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: Edwards Scancoat 6, 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV
Details: 2.9 mM fluorinated Fos-choline 8 was added prior to sample application onto grids. Grids were blotted for 3.5-4 sec..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 1906 / Average exposure time: 9.0 sec. / Average electron dose: 50.1 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 1.8 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 48924 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 154619
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated with cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 54660
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2)
FSC plot (resolution estimation)

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