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

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Basic information

Entry
Database: PDB / ID: 7nnu
TitleCryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs
Components
  • Conserved hypothetical membrane protein
  • Energy-coupling factor transporter ATP-binding protein EcfA1
  • Energy-coupling factor transporter ATP-binding protein EcfA2
  • Energy-coupling factor transporter transmembrane protein EcfT
KeywordsTRANSPORT PROTEIN / ABC Transporter / Type III ABC Transporter / ECF transporter complex / Folate transporter / 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 / 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 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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

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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: Conserved hypothetical membrane protein
D: Energy-coupling factor transporter transmembrane protein EcfT


Theoretical massNumber of molelcules
Total (without water)115,6124
Polymers115,6124
Non-polymers00
Water63135
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12950 Å2
ΔGint-113 kcal/mol
Surface area40030 Å2
MethodPISA

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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 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
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 35 / Source method: isolated from a natural source / Formula: H2O

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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 (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
SpecimenConc.: 7.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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: 2.9 mM fluorinated Fos-choline 8 was added prior to sample application onto grids. Grids were blotted for 3-4 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 FIELDBright-field microscopy / Nominal magnification: 165000 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: 51.1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 4722
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 7676 / Height: 7420 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.18_3861: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.5.0particle selectionPhosaurusNet architecture
2EPU2.4image acquisition
4CTFFIND4.1.13CTF correction
7Coot0.9-premodel fitting
9RELION3.1-betainitial Euler assignment
10RELION3.1-betafinal Euler assignment
12RELION3.1-beta3D reconstruction
13PHENIX1.18-3861model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1362547
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166524 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
17NNT

7nnt

A1
27NNT

7nnt

B1
37NNT

7nnt

C1
47NNT

7nnt

D1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0077850
ELECTRON MICROSCOPYf_angle_d0.78510630
ELECTRON MICROSCOPYf_dihedral_angle_d12.3891038
ELECTRON MICROSCOPYf_chiral_restr0.0541229
ELECTRON MICROSCOPYf_plane_restr0.0071327

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