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

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Basic information

Entry
Database: PDB / ID: 8bmq
TitleCryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs bound to AMP-PNP
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 / AMP-PNP
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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / 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: 3.6 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
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)116,6497
Polymers115,6124
Non-polymers1,0373
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14730 Å2
ΔGint-118 kcal/mol
Surface area42680 Å2
MethodPISA

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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 AMP-PNP
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 AMP-PNP 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 , 2 types, 3 molecules

#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: 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.: 6.2 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-AMP-PNP 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-AMP-PNP 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: 1 / Num. of real images: 4052
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
2SerialEM3.8.0 betaimage acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.20.1-4487-000model refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 854709
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129663 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057949
ELECTRON MICROSCOPYf_angle_d0.59610781
ELECTRON MICROSCOPYf_dihedral_angle_d6.8631063
ELECTRON MICROSCOPYf_chiral_restr0.0441247
ELECTRON MICROSCOPYf_plane_restr0.0041336

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