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- EMDB-16120: Cryo-EM structure of the folate-specific ECF transporter complex ... -

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Entry
Database: EMDB / ID: EMD-16120
TitleCryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs bound to ATP and ADP
Map dataInward-facing apo conformation of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs bound to ATP and ADP at 3.2 A resolution sharpened at -113 A^2.
Sample
  • Complex: Folate-specific ECF transporter complex
    • 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: Folate family ECF transporter S component
    • Protein or peptide: Energy-coupling factor transporter transmembrane protein EcfT
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsABC Transporter / ECF transporter complex / ATP / ADP / 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 / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
ECF transporter S component, folate family / ECF transporter, substrate-specific component / ECF transporter transmembrane protein EcfT / ECF transporter, substrate-specific component / 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 ...ECF transporter S component, folate family / ECF transporter, substrate-specific component / ECF transporter transmembrane protein EcfT / ECF transporter, substrate-specific component / 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
Conserved hypothetical membrane protein / 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: 3.2 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_16120.png

Downloads & links

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Map

FileDownload / File: emd_16120.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInward-facing apo conformation of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs bound to ATP and ADP at 3.2 A resolution sharpened at -113 A^2.
Voxel sizeX=Y=Z: 1.022 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-2.6763203 - 4.8570213
Average (Standard dev.)0.008841563 (±0.10267058)
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_16120_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 model...

Fileemd_16120_additional_1.map
AnnotationSharpened map obtained with DeepEMhancer used for model building and figures.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_16120_half_map_1.map
AnnotationHalf map 2 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_16120_half_map_2.map
AnnotationHalf map 1 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Folate-specific ECF transporter complex

EntireName: Folate-specific ECF transporter complex
Components
  • Complex: Folate-specific ECF transporter complex
    • 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: Folate family ECF transporter S component
    • Protein or peptide: Energy-coupling factor transporter transmembrane protein EcfT
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Folate-specific ECF transporter complex

SupramoleculeName: Folate-specific ECF transporter complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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 / Details: Bound to ATP / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Molecular weightTheoretical: 33.166418 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MHHHHHHHHH HGENLYFQGS DNIISFDHVT FTYPDSPRPA LSDLSFAIER GSWTALIGHN GSGKSTVSKL INGLLAPDDL DKSSITVDG VKLGADTVWE VREKVGIVFQ NPDNQFVGAT VSDDVAFGLE NRAVPRPEML KIVAQAVADV GMADYADSEP S NLSGGQKQ ...String:
MHHHHHHHHH HGENLYFQGS DNIISFDHVT FTYPDSPRPA LSDLSFAIER GSWTALIGHN GSGKSTVSKL INGLLAPDDL DKSSITVDG VKLGADTVWE VREKVGIVFQ NPDNQFVGAT VSDDVAFGLE NRAVPRPEML KIVAQAVADV GMADYADSEP S NLSGGQKQ RVAIAGILAV KPQVIILDES TSMLDPEGKE QILDLVRKIK EDNNLTVISI THDLEEAAGA DQVLVLDDGQ LL DQGKPEE IFPKVEMLKR IGLDIPFVYR LKQLLKERGI VLPDEIDDDE KLVQSLWQLN SKM

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 / Details: Bound to ADP and Mg / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Molecular weightTheoretical: 31.672156 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MAIKFENVSY VYSPGSPLEA IGLDQLNFSL EEGKFIALVG HTGSGKSTLM QHFNALLKPT SGKIEIAGYT ITPETGNKGL KDLRRKVSL AFQFSEAQLF ENTVLKDVEY GPRNFGFSED EAREAALKWL KKVGLKDDLI EHSPFDLSGG QMRRVALAGV L AYEPEIIC ...String:
MAIKFENVSY VYSPGSPLEA IGLDQLNFSL EEGKFIALVG HTGSGKSTLM QHFNALLKPT SGKIEIAGYT ITPETGNKGL KDLRRKVSL AFQFSEAQLF ENTVLKDVEY GPRNFGFSED EAREAALKWL KKVGLKDDLI EHSPFDLSGG QMRRVALAGV L AYEPEIIC LDEPAAGLDP MGRLEMMQLF KDYQAAGHTV ILVTHNMDDV ADYADDVLAL EHGRLIKHAS PKEVFKDSEW LQ KHHLAEP RSARFAAKLE AAGLKLPGQP LTMPELADAI KQSLKGGEHE

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

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Macromolecule #3: Folate family ECF transporter S component

MacromoleculeName: Folate family ECF transporter S component / type: protein_or_peptide / ID: 3 / Number of copies: 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
Molecular weightTheoretical: 20.483604 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
MKSESKVSSK LELRELVLLA MVIAIKVILG QFKVGNATLQ VGLGFIGSVM LGYLFGPWWG FAGGALSDLV SSVIFGNLGG FFIGFTLTA ALGPMIYGFF LYKQPIQIWR VIASVICVTV ICNIGLNTLW VSMMYGINFM VALSSRILKE MITPWIQMVA V WFILEGLS RVKLSRKFWS HPQFEK

UniProtKB: Conserved hypothetical membrane protein

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Macromolecule #4: Energy-coupling factor transporter transmembrane protein EcfT

MacromoleculeName: Energy-coupling factor transporter transmembrane protein EcfT
type: protein_or_peptide / ID: 4 / Number of copies: 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
Molecular weightTheoretical: 30.290283 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MSKIIIGRYL PGTTFVYRVD PRAKLLTTFY FIIMIFLANN WVSYLVISIF GLAYVFATGL KARVFWDGVK PMIWMIVFTS LLQTFFMAG GKVYWHWWIF TLSSEGLING LYVFIRFAMI ILVSTVMTVT TKPLEIADAM EWMLTPLKLF KVNVGMISLV I SIALRFVP ...String:
MSKIIIGRYL PGTTFVYRVD PRAKLLTTFY FIIMIFLANN WVSYLVISIF GLAYVFATGL KARVFWDGVK PMIWMIVFTS LLQTFFMAG GKVYWHWWIF TLSSEGLING LYVFIRFAMI ILVSTVMTVT TKPLEIADAM EWMLTPLKLF KVNVGMISLV I SIALRFVP TLFDQTVKIM NAQRSRGADF NDGGLVKRAK SVVPMLVPLF IDSLEVALDL STAMESRGYK GSEGRTRYRI LE WSKVDLI PVAYCLLLTI LMITTRKH

UniProtKB: Energy-coupling factor transporter transmembrane protein EcfT

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration7.4 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: 5 mM Mg-ATP and 10 uM folate were added to the concentrated sample and incubated at 37 degC for 10 min. 2.9 mM fluorinated Fos-choline 8 was added prior to sample application onto grids. ...Details: 5 mM Mg-ATP and 10 uM folate were added to the concentrated sample and incubated at 37 degC for 10 min. 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
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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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: 2 / Number real images: 3286 / Average exposure time: 9.0 sec. / Average electron dose: 50.1 e/Å2
Details: Dataset 1: 502 movies (collected with EPU from grid 1) Dataset 2: 597 movies (collected with EPU from grid 2) Dataset 3: 2187 movies (collected with SerialEM from grid 2)
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1207115
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated with cryoSPARC
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 167227
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8bmp:
Cryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs bound to ATP and ADP

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