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- EMDB-12690: ABC transporter NosDFY, nucleotide-free in lipid nanodisc, R-domain 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-12690
TitleABC transporter NosDFY, nucleotide-free in lipid nanodisc, R-domain 2
Map dataNanodisc-subtracted refinement,R-domain conformation 2, unsharpened
Sample
  • Complex: ABC Transporter NosFY in complex with accessory periplasmic protein NosD
    • 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
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsABC Transporter complex / nucleotide-free / MEMBRANE PROTEIN
Function / homology
Function and homology information


ABC-type transporter activity / periplasmic space / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
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 ...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. / 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
Similarity search - Component
Biological speciesPseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Methodsingle particle reconstruction / cryo EM
AuthorsMueller C / Zhang L
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
DepositionMar 28, 2021-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_12690.png

Downloads & links

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Map

FileDownload / File: emd_12690.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNanodisc-subtracted refinement,R-domain conformation 2, unsharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 200 pix.
= 259.2 Å		1.3 Å/pix.
x 200 pix.
= 259.2 Å		1.3 Å/pix.
x 200 pix.
= 259.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.296 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.036594518 - 0.06785158
Average (Standard dev.)-0.000023808001 (±0.0031769495)
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: Nanodisc-subtracted refinement,R-domain conformation 2, sharpened

Fileemd_12690_additional_1.map
AnnotationNanodisc-subtracted refinement,R-domain conformation 2, sharpened
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
Components
  • Complex: ABC Transporter NosFY in complex with accessory periplasmic protein NosD
    • 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
  • Ligand: MAGNESIUM 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
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Nucleotide-free state in lipid nanodisc, R-domain conformation 2
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (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

UniProtKB: Probable ABC transporter binding protein NosD

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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

UniProtKB: Probable ABC transporter ATP-binding protein NosF

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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

UniProtKB: Probable ABC transporter permease protein NosY

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration15 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Average exposure time: 1.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber images used: 138319
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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