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- PDB-8bmp: Cryo-EM structure of the folate-specific ECF transporter complex ... -

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Basic information

Entry
Database: PDB / ID: 8bmp
TitleCryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs bound to ATP and ADP
Components
  • (Energy-coupling factor transporter ATP-binding protein ...) x 2
  • Energy-coupling factor transporter transmembrane protein EcfT
  • Folate family ECF transporter S component
KeywordsMEMBRANE PROTEIN / ABC Transporter / ECF transporter complex / ATP / ADP
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 / 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
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsThangaratnarajah, C. / Rheinberger, J. / Paulino, C. / Slotboom, D.J.
Funding supportEuropean Union, Netherlands, 5items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 9, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Energy-coupling factor transporter ATP-binding protein EcfA1
B: Energy-coupling factor transporter ATP-binding protein EcfA2
C: Folate family ECF transporter S component
D: Energy-coupling factor transporter transmembrane protein EcfT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)116,5717
Polymers115,6124
Non-polymers9593
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14470 Å2
ΔGint-132 kcal/mol
Surface area42480 Å2

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Components

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Energy-coupling factor transporter ATP-binding protein ... , 2 types, 2 molecules AB

#1: Protein Energy-coupling factor transporter ATP-binding protein EcfA1 / ECF transporter A component EcfA1


Mass: 33166.418 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Bound to ATP
Source: (gene. exp.) Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Gene: ecfA1, cbiO1, Ldb0424 / Plasmid: p2BAD / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: Q1GBJ0, Translocases
#2: Protein Energy-coupling factor transporter ATP-binding protein EcfA2 / ECF transporter A component EcfA2


Mass: 31672.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Bound to ADP and Mg
Source: (gene. exp.) Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Gene: ecfA2, cbiO2, Ldb0425 / Plasmid: p2BAD / Production host: Escherichia coli MC1061 (bacteria)
References: UniProt: Q1GBI9, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances

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Protein , 2 types, 2 molecules CD

#3: Protein Folate family ECF transporter S component / Conserved hypothetical membrane protein


Mass: 20483.604 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Gene: Ldb1625 / Plasmid: p2BAD / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: Q1G929
#4: Protein Energy-coupling factor transporter transmembrane protein EcfT / ECF transporter T component EcfT


Mass: 30290.283 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Gene: cbiQ, ecfT, Ldb0426 / Plasmid: p2BAD / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: Q1GBI8

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Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

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: Folate-specific ECF transporter complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)
Source (recombinant)Organism: Escherichia coli MC1061 (bacteria) / Plasmid: p2BAD
Buffer solutionpH: 8 / Details: 20 mM Tris, pH 8.0, 150 mM NaCl
SpecimenConc.: 7.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Edwards Scancoat 6, 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288 K
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 imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 48924 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 50.1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3286
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)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.7.5particle selection
2EPU2.7.0image acquisition
3SerialEM3.8.0 betaimage acquisition
5CTFFIND4.1.14CTF correction
8Coot0.9.8.1model fitting
10PHENIX1.20.1-4487-000model refinement
11cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.2final Euler assignment
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1207115
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 167227 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037961
ELECTRON MICROSCOPYf_angle_d0.57510797
ELECTRON MICROSCOPYf_dihedral_angle_d5.6641060
ELECTRON MICROSCOPYf_chiral_restr0.0431243
ELECTRON MICROSCOPYf_plane_restr0.0041341

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