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- EMDB-16122: Cryo-EM structure of the wild-type solitary ECF module in MSP2N2 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-16122
TitleCryo-EM structure of the wild-type solitary ECF module in MSP2N2 lipid nanodiscs in the ATPase open and nucleotide-free conformation
Map dataATPase open and nucleotide-free conformation of the wild-type solitary ECF module in MSP2N2 lipid nanodiscs at 3.8 A resolution sharpened at -148 A^2.
Sample
  • Complex: Wild-type solitary ECF module
    • 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: Energy-coupling factor transporter transmembrane protein EcfT
KeywordsABC Transporter / ECF transporter complex / motor / 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 / plasma membrane
Similarity search - Function
ECF transporter transmembrane protein EcfT / 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 ...ECF transporter transmembrane protein EcfT / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsThangaratnarajah C / Rheinberger J / Paulino C / Slotboom DJ
Funding supportEuropean Union, Netherlands, 5 items
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, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16122.png

Downloads & links

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Map

FileDownload / File: emd_16122.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationATPase open and nucleotide-free conformation of the wild-type solitary ECF module in MSP2N2 lipid nanodiscs at 3.8 A resolution sharpened at -148 A^2.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 256 pix.
= 261.632 Å		1.02 Å/pix.
x 256 pix.
= 261.632 Å		1.02 Å/pix.
x 256 pix.
= 261.632 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.022 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.475
Minimum - Maximum-2.585929 - 3.861902
Average (Standard dev.)0.0043650456 (±0.078004986)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 261.632 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16122_msk_1.map
Projections & Slices
AxesZYX

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

Fileemd_16122_additional_1.map
AnnotationSharpened map obtained with DeepEMhancer used for model building and figures.
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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

Fileemd_16122_half_map_1.map
AnnotationHalf map 2 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 1 used during refinement and FSC...

Fileemd_16122_half_map_2.map
AnnotationHalf map 1 used during refinement and FSC gold-standard resolution calculation.
Projections & Slices
AxesZYX

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Sample components

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Entire : Wild-type solitary ECF module

EntireName: Wild-type solitary ECF module
Components
  • Complex: Wild-type solitary ECF module
    • 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: Energy-coupling factor transporter transmembrane protein EcfT

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Supramolecule #1: Wild-type solitary ECF module

SupramoleculeName: Wild-type solitary ECF module / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (bacteria)

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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 ATCC 11842 = JCM 1002 (bacteria)
Strain: ATCC 11842 / DSM 20081 / BCRC 10696 / JCM 1002 / NBRC 13953 / NCIMB 11778 / NCTC 12712 / WDCM 00102 / Lb 14
Molecular weightTheoretical: 30.801861 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: GSDNIISFDH VTFTYPDSPR PALSDLSFAI ERGSWTALIG HNGSGKSTVS KLINGLLAPD DLDKSSITVD GVKLGADTVW EVREKVGIV FQNPDNQFVG ATVSDDVAFG LENRAVPRPE MLKIVAQAVA DVGMADYADS EPSNLSGGQK QRVAIAGILA V KPQVIILD ...String:
GSDNIISFDH VTFTYPDSPR PALSDLSFAI ERGSWTALIG HNGSGKSTVS KLINGLLAPD DLDKSSITVD GVKLGADTVW EVREKVGIV FQNPDNQFVG ATVSDDVAFG LENRAVPRPE MLKIVAQAVA DVGMADYADS EPSNLSGGQK QRVAIAGILA V KPQVIILD ESTSMLDPEG KEQILDLVRK IKEDNNLTVI SITHDLEEAA GADQVLVLDD GQLLDQGKPE EIFPKVEMLK RI GLDIPFV YRLKQLLKER GIVLPDEIDD DEKLVQSLWQ LNSKM

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 ATCC 11842 = JCM 1002 (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: Energy-coupling factor transporter transmembrane protein EcfT

MacromoleculeName: Energy-coupling factor transporter transmembrane protein EcfT
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 = JCM 1002 (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

Concentration5.4 mg/mL
BufferpH: 8 / Details: 20 mM Tris, pH 8.0, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: Edwards Scancoat 6, 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 3.5-4 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 / Calibrated defocus max: 1.8 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 48924 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
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: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 4 / Number real images: 12071 / Average exposure time: 9.0 sec. / Average electron dose: 50.1 e/Å2
Details: Dataset 1: 2200 movies Dataset 2: 4608 movie Dataset 3: 2101 movie Dataset 4: 3162 movie
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2889054
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated with cryoSPARC
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 93755
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8bmr:
Cryo-EM structure of the wild-type solitary ECF module in MSP2N2 lipid nanodiscs in the ATPase open and nucleotide-free conformation

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