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- EMDB-12484: Cryo-EM structure of the folate-specific ECF transporter complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12484
TitleCryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs
Map dataInward-facing apo conformation of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs at 2.7 A resolution sharpened at -44 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: Conserved hypothetical membrane protein
    • Protein or peptide: Energy-coupling factor transporter transmembrane protein EcfT
  • Ligand: water
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 / 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 (strain ATCC 11842 / DSM 20081 / JCM 1002 / NBRC 13953 / NCIMB 11778) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsThangaratnarajah C / Rheinberger J / Paulino C / Slotboom DJ
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-
UpdateSep 22, 2021-
Current statusSep 22, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0209
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0209
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nnu
  • Surface level: 0.0209
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12484.png

Downloads & links

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Map

FileDownload / File: emd_12484.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInward-facing apo conformation of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs at 2.7 A resolution sharpened at -44 A^2.
Voxel sizeX=Y=Z: 0.771 Å
Density
Contour LevelBy AUTHOR: 0.0209 / Movie #1: 0.0209
Minimum - Maximum-0.08072819 - 0.13810913
Average (Standard dev.)3.075442e-06 (±0.0034847322)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 296.06403 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.7710.7710.771
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z296.064296.064296.064
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ540540540
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0810.1380.000

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Supplemental data

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Mask #1

Fileemd_12484_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Sharpened map obtained with LocalDeblur used for model...

Fileemd_12484_additional_1.map
AnnotationSharpened map obtained with LocalDeblur used for model building and figures.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_12484_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_12484_half_map_2.map
AnnotationHalf map 2 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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: Conserved hypothetical membrane protein
    • Protein or peptide: Energy-coupling factor transporter transmembrane protein EcfT
  • Ligand: water

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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: #1-#4
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
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria) / Recombinant plasmid: p2BAD

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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

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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

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Macromolecule #3: Conserved hypothetical membrane protein

MacromoleculeName: Conserved hypothetical membrane protein / 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

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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

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 35 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 3 / Number real images: 4722 / Average exposure time: 9.0 sec. / Average electron dose: 51.1 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1362547
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Startup modelType of model: INSILICO MODEL
In silico model: Initial model generated by the stochastic gradient decent (SGD) method.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1-beta)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1-beta)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1-beta) / Number images used: 166524
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7nnt

chain_id: A

7nnt

chain_id: B

7nnt

chain_id: C

7nnt

chain_id: D
RefinementSpace: REAL
Output model

PDB-7nnu:
Cryo-EM structure of the folate-specific ECF transporter complex in MSP2N2 lipid nanodiscs

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