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- EMDB-12483: Cryo-EM structure of the folate-specific ECF transporter complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12483
TitleCryo-EM structure of the folate-specific ECF transporter complex in DDM micelles
Map dataInward-facing apo conformation of the folate-specific ECF transporter complex in DDM micelles at 3.4 A resolution sharpened at -62 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
KeywordsABC Transporter / Type III ABC Transporter / ECF transporter complex / Folate transporter / Membrane protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


Translocases / 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. / : ...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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsThangaratnarajah C / Rheinberger J
Funding support Netherlands, 4 items
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, 2021-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0329
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0329
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nnt
  • Surface level: 0.0329
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12483.png

Downloads & links

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Map

FileDownload / File: emd_12483.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 DDM micelles at 3.4 A resolution sharpened at -62 A^2.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 259.072 Å		1.01 Å/pix.
x 256 pix.
= 259.072 Å		1.01 Å/pix.
x 256 pix.
= 259.072 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.012 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0329 / Movie #1: 0.0329
Minimum - Maximum-0.11336199 - 0.18909028
Average (Standard dev.)-0.0000034140137 (±0.005476082)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 259.072 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z259.072259.072259.072
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ540540540
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1130.189-0.000

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Supplemental data

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Mask #1

Fileemd_12483_msk_1.map
Projections & Slices
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Additional map: Sharpened map obtained with LocalDeblur used for model...

Fileemd_12483_additional_1.map
AnnotationSharpened map obtained with LocalDeblur used for model building and figures.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_12483_half_map_1.map
AnnotationHalf map 1 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_12483_half_map_2.map
AnnotationHalf map 2 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

Projections

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

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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: all
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

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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 / Number of copies: 1 / Enantiomer: LEVO / EC number: Translocases
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
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 / 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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7.9 mg/mL
BufferpH: 8
Details: 20 mM Tris, pH 8.0, 150 mM NaCl, 0.0261 % (w/v) DDM
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: 5 mA; for dataset 2.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV
Details: Grid was blotted for 4 sec after a wait time for 2.5 sec..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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: 823 / Average exposure time: 9.0 sec. / Average electron dose: 53.3 e/Å2
Details: Dataset 1: 466 movies (Quantifoil grid) Dataset 2: 357 movies (UltrAuFoil grid)
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 161463
Startup modelType of model: INSILICO MODEL
In silico model: Initial model generated by the stochastic gradient decent (SGD) method.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number images used: 41963
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0.7)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0.7)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5jsz

chain_id: A, source_name: PDB, initial_model_type: experimental model

5jsz

chain_id: B, source_name: PDB, initial_model_type: experimental model

5jsz

chain_id: C, source_name: PDB, initial_model_type: experimental model

5jsz

chain_id: D, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL
Output model

PDB-7nnt:
Cryo-EM structure of the folate-specific ECF transporter complex in DDM micelles

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