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- PDB-7nnt: Cryo-EM structure of the folate-specific ECF transporter complex ... -

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Basic information

Entry
Database: PDB / ID: 7nnt
TitleCryo-EM structure of the folate-specific ECF transporter complex in DDM micelles
Components
  • Energy-coupling factor transporter ATP-binding protein EcfA1
  • Energy-coupling factor transporter ATP-binding protein EcfA2
  • Energy-coupling factor transporter transmembrane protein EcfT
  • Folate family ECF transporter S component
KeywordsTRANSPORT PROTEIN / ABC Transporter / Type III ABC Transporter / ECF transporter complex / Folate transporter / Membrane protein
Function / homology
Function and homology information


Translocases / ATPase-coupled transmembrane transporter activity / 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. / : ...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 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsThangaratnarajah, C. / Rheinberger, J. / Paulino, C. / Slotboom, D.J.
Funding support Netherlands, 4items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)714.018.003 Netherlands
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
European Union (EU)847675 Netherlands
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Insights into the bilayer-mediated toppling mechanism of a folate-specific ECF transporter by cryo-EM.
Authors: Chancievan Thangaratnarajah / Jan Rheinberger / Cristina Paulino / Dirk J Slotboom /
Abstract: Energy-coupling factor (ECF)-type transporters are small, asymmetric membrane protein complexes (∼115 kDa) that consist of a membrane-embedded, substrate-binding protein (S component) and a ...Energy-coupling factor (ECF)-type transporters are small, asymmetric membrane protein complexes (∼115 kDa) that consist of a membrane-embedded, substrate-binding protein (S component) and a tripartite ATP-hydrolyzing module (ECF module). They import micronutrients into bacterial cells and have been proposed to use a highly unusual transport mechanism, in which the substrate is dragged across the membrane by a toppling motion of the S component. However, it remains unclear how the lipid bilayer could accommodate such a movement. Here, we used cryogenic electron microscopy at 200 kV to determine structures of a folate-specific ECF transporter in lipid nanodiscs and detergent micelles at 2.7- and 3.4-Å resolution, respectively. The structures reveal an irregularly shaped bilayer environment around the membrane-embedded complex and suggest that toppling of the S component is facilitated by protein-induced membrane deformations. In this way, structural remodeling of the lipid bilayer environment is exploited to guide the transport process.
History
DepositionFeb 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 22, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Structure viewerMolecule:
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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


Theoretical massNumber of molelcules
Total (without water)115,6124
Polymers115,6124
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#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
Source: (gene. exp.) Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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
Source: (gene. exp.) Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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
#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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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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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: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (bacteria)
Strain: ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778
Source (recombinant)Organism: Escherichia coli MC1061 (bacteria) / Plasmid: p2BAD
Buffer solutionpH: 8
Details: 20 mM Tris, pH 8.0, 150 mM NaCl, 0.0261 % (w/v) DDM
SpecimenConc.: 7.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
IDSpecimen-IDDetailsGrid materialGrid type
115 mA; for dataset 1.GOLDQuantifoil R1.2/1.3
215 mA; for dataset 2.GOLDUltrAuFoil R1.2/1.3
Vitrification

Specimen-ID: 1 / Chamber temperature: 288 K / Cryogen name: ETHANE-PROPANE / Details: Grid was blotted for 4 sec after a wait time for 2.5 sec. / Entry-ID: 7NNT / Humidity: 100 % / Instrument: FEI VITROBOT MARK IV

ID
1
2

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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 / 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: 53.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 823
Details: Dataset 1: 466 movies (Quantifoil grid) Dataset 2: 357 movies (UltrAuFoil grid)
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: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPU1.12image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.9-premodel fitting
9PHENIX1.18-3861model refinement
10RELION3.0.7initial Euler assignment
11RELION3.0.7final Euler assignment
13RELION3.0.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 161463
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41963 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 5JSZ / Initial refinement model-ID: 1 / PDB-ID: 5JSZ

5jsz

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3C
4D
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057827
ELECTRON MICROSCOPYf_angle_d0.61510599
ELECTRON MICROSCOPYf_dihedral_angle_d12.0881035
ELECTRON MICROSCOPYf_chiral_restr0.0461223
ELECTRON MICROSCOPYf_plane_restr0.0051325

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