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- EMDB-13050: ABC Transporter complex NosDFYL, consensus refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-13050
TitleABC Transporter complex NosDFYL, consensus refinement
Map dataunsharpened map
Sample
  • Complex: ABC Transporter NosFY in complex with accessory periplasmic protein NosD and Cu chaperone NosL
    • Protein or peptide: Probable ABC transporter binding protein NosD
    • Protein or peptide: Probable ABC transporter ATP-binding protein NosF
    • Protein or peptide: Probable ABC transporter permease protein NosY
    • Protein or peptide: Copper-binding lipoprotein NosL
  • Ligand: MAGNESIUM ION
  • Ligand: COPPER (II) ION
  • Ligand: ZINC ION
  • Ligand: water
Function / homology
Function and homology information


ABC-type transporter activity / periplasmic space / ATP hydrolysis activity / ATP binding / metal ion binding / plasma membrane
Similarity search - Function
Nitrous oxide reductase accessory protein NosL / NosL / Nitrous oxide reductase family maturation protein NosD / ABC-2 family transporter protein / ABC-2 family transporter protein / Parallel beta-helix repeat-2 / Periplasmic copper-binding protein NosD, beta helix domain / Carbohydrate-binding/sugar hydrolysis domain / Domain present in carbohydrate binding proteins and sugar hydrolses / Periplasmic copper-binding protein (NosD) ...Nitrous oxide reductase accessory protein NosL / NosL / Nitrous oxide reductase family maturation protein NosD / ABC-2 family transporter protein / ABC-2 family transporter protein / Parallel beta-helix repeat-2 / Periplasmic copper-binding protein NosD, beta helix domain / Carbohydrate-binding/sugar hydrolysis domain / Domain present in carbohydrate binding proteins and sugar hydrolses / Periplasmic copper-binding protein (NosD) / Parallel beta-helix repeat / Parallel beta-helix repeats / Pectin lyase fold / Pectin lyase fold/virulence factor / 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. / Prokaryotic membrane lipoprotein lipid attachment site profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Probable ABC transporter binding protein NosD / Probable ABC transporter ATP-binding protein NosF / Probable ABC transporter permease protein NosY / Copper-binding lipoprotein NosL
Similarity search - Component
Biological speciesPseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMueller C / Zhang L / Lu W / Du J / Einsle O
CitationJournal: Nature / Year: 2022
Title: Molecular interplay of an assembly machinery for nitrous oxide reductase.
Authors: Christoph Müller / Lin Zhang / Sara Zipfel / Annika Topitsch / Marleen Lutz / Johannes Eckert / Benedikt Prasser / Mohamed Chami / Wei Lü / Juan Du / Oliver Einsle /
Abstract: Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (NO) from soils and industrial processes have increased considerably over the last decades. As the final step of bacterial ...Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (NO) from soils and industrial processes have increased considerably over the last decades. As the final step of bacterial denitrification, NO is reduced to chemically inert N (refs. ) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (NOR) (ref. ). The assembly of its unique [4Cu:2S] active site cluster Cu requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. ). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and NOR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to NOR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with NOR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme NOR.
History
DepositionJun 8, 2021-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13050.png

Downloads & links

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Map

FileDownload / File: emd_13050.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Voxel sizeX=Y=Z: 1.296 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.035266124 - 0.09057219
Average (Standard dev.)-4.8169935e-05 (±0.0034378003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened map

Fileemd_13050_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ABC Transporter NosFY in complex with accessory periplasmic prote...

EntireName: ABC Transporter NosFY in complex with accessory periplasmic protein NosD and Cu chaperone NosL
Components
  • Complex: ABC Transporter NosFY in complex with accessory periplasmic protein NosD and Cu chaperone NosL
    • Protein or peptide: Probable ABC transporter binding protein NosD
    • Protein or peptide: Probable ABC transporter ATP-binding protein NosF
    • Protein or peptide: Probable ABC transporter permease protein NosY
    • Protein or peptide: Copper-binding lipoprotein NosL
  • Ligand: MAGNESIUM ION
  • Ligand: COPPER (II) ION
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: ABC Transporter NosFY in complex with accessory periplasmic prote...

SupramoleculeName: ABC Transporter NosFY in complex with accessory periplasmic protein NosD and Cu chaperone NosL
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: nucleotide-free state
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Probable ABC transporter binding protein NosD

MacromoleculeName: Probable ABC transporter binding protein NosD / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Molecular weightTheoretical: 48.2585 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MFKAQATFSR YSAAVSLLLL FSGAAQAAPQ SITTLPLQPD GENRWRLPAG EYQGQFTIEQ PMQLRCEPGA VIQSQGQGSS LLISAPDVL VEGCTLYEWG SDLTAMDSAV FILPAAERAQ ISNNRMRGPG FGVFVDGTRD VQVIGNEIDG DAGVRSQDRG N GIHLFAVS ...String:
MFKAQATFSR YSAAVSLLLL FSGAAQAAPQ SITTLPLQPD GENRWRLPAG EYQGQFTIEQ PMQLRCEPGA VIQSQGQGSS LLISAPDVL VEGCTLYEWG SDLTAMDSAV FILPAAERAQ ISNNRMRGPG FGVFVDGTRD VQVIGNEIDG DAGVRSQDRG N GIHLFAVS GARVLHNHVR NARDGIYIDT SNGNHLEGNV IEDVRYGVHY MFANENSLID NVTRRTRTGY ALMQSRKLTV TG NRSEQDQ NYGILMNYIT YSTITGNFVS DVQRGDTGGD SMISGGEGKA LFIYNSLFNT IENNHFEKSS LGIHLTAGSE DNR ISGNAF VGNQQQVKYV ASRTQEWSVD GRGNYWSDYL GWDRNNDGLG DIAYEPNDNV DRLLWLYPQV RLLMNSPSIE VLRW VQRAF PVIKSPGVQD SHPLMKLPTE KLLTEKQEPT S

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Macromolecule #2: Probable ABC transporter ATP-binding protein NosF

MacromoleculeName: Probable ABC transporter ATP-binding protein NosF / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Molecular weightTheoretical: 33.821703 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MNAVEIQGVS QRYGSMTVLH DLNLNLGEGE VLGLFGHNGA GKTTSMKLIL GLLSPSEGQV KVLGRAPNDP QVRRQLGYLP ENVTFYPQL SGRETLRHFA RLKGAALTQV DELLEQVGLA HAADRRVKTY SKGMRQRLGL AQALLGEPRL LLLDEPTVGL D PIATQDLY ...String:
MNAVEIQGVS QRYGSMTVLH DLNLNLGEGE VLGLFGHNGA GKTTSMKLIL GLLSPSEGQV KVLGRAPNDP QVRRQLGYLP ENVTFYPQL SGRETLRHFA RLKGAALTQV DELLEQVGLA HAADRRVKTY SKGMRQRLGL AQALLGEPRL LLLDEPTVGL D PIATQDLY LLIDRLRQRG TSIILCSHVL PGVEAHINRA AILAKGCLQA VGSLSQLRAE AGLPVRIRAS GISERDSWLQ RW TDAGHSA RGLSESSIEV VAVNGHKLVL LRQLLGEGEP EDIEIHQPSL EDLYRYYMER AGDVRAQEGR L

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Macromolecule #3: Probable ABC transporter permease protein NosY

MacromoleculeName: Probable ABC transporter permease protein NosY / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Molecular weightTheoretical: 29.449203 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MNQVWNIARK ELSDGLRNRW LLAISLLFAV LAVGIAWLGA AASGQLGFTS IPATIASLAS LATFLMPLIA LLLAYDAIVG EDEGGTLML LLTYPLGRGQ ILLGKFVGHG LILALAVLIG FGCAALAIAL LVEGVELGML FWAFGRFMIS STLLGWVFLA F AYVLSGKV ...String:
MNQVWNIARK ELSDGLRNRW LLAISLLFAV LAVGIAWLGA AASGQLGFTS IPATIASLAS LATFLMPLIA LLLAYDAIVG EDEGGTLML LLTYPLGRGQ ILLGKFVGHG LILALAVLIG FGCAALAIAL LVEGVELGML FWAFGRFMIS STLLGWVFLA F AYVLSGKV NEKSSAAGLA LGVWFLFVLV FDLVLLALLV LSEGKFNPEL LPWLLLLNPT DIYRLINLSG FEGSGSAMGV LS LGADLPV PAAVLWLCLL AWIGVSLLLA YAIFRRRLT

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Macromolecule #4: Copper-binding lipoprotein NosL

MacromoleculeName: Copper-binding lipoprotein NosL / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Molecular weightTheoretical: 20.47015 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
MNALHRIGAG TLLAVLLAFG LTGCGEKEEV QQSLEPVAFH DSDECHVCGM IITDFPGPKG QAVEKRGVKK FCSTAEMLGW WLQPENRLL DAKLYVHDMG RSVWEKPDDG HLIDATSAYY VVGTSLKGAM GASLASFAEE QDAKALAGMH GGRVLRFEEI D QALLQEAA SMQHGGMHDH APNGAHNAHA GH

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: COPPER (II) ION

MacromoleculeName: COPPER (II) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: CU
Molecular weightTheoretical: 63.546 Da
Chemical component information

ChemComp-CU:
COPPER (II) ION / Copper

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 1 / 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

Concentration12 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 165858
FSC plot (resolution estimation)

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