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- EMDB-13885: CryoEM structure of the ABC transporter NosDFY complexed with nit... -

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Basic information

Entry
Database: EMDB / ID: EMD-13885
TitleCryoEM structure of the ABC transporter NosDFY complexed with nitrous oxide reductase NosZ
Map datadensity modified map using Phenix
Sample
  • Complex: Heteroheptamer complex of NosZDFY
    • 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: Nitrous-oxide reductase
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


nitrous-oxide reductase / nitrous-oxide reductase activity / copper ion import / denitrification pathway / cytochrome-c oxidase activity / ABC-type transporter activity / periplasmic space / copper ion binding / calcium ion binding / ATP hydrolysis activity ...nitrous-oxide reductase / nitrous-oxide reductase activity / copper ion import / denitrification pathway / cytochrome-c oxidase activity / ABC-type transporter activity / periplasmic space / copper ion binding / calcium ion binding / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
Nitrous-oxide reductase / Nitrous-oxide reductase, C-terminal / Nitrous oxide reductase, propeller repeat 1 / Nitrous oxide reductase, propeller repeat 2 / Nitrous oxide reductase propeller repeat / Nitrous oxide reductase propeller repeat 2 / Nitrous oxide reductase family maturation protein NosD / ABC-2 family transporter protein / ABC-2 family transporter protein / Parallel beta-helix repeat-2 ...Nitrous-oxide reductase / Nitrous-oxide reductase, C-terminal / Nitrous oxide reductase, propeller repeat 1 / Nitrous oxide reductase, propeller repeat 2 / Nitrous oxide reductase propeller repeat / Nitrous oxide reductase propeller repeat 2 / 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 / Nitrous oxide reductase, N-terminal / Domain present in carbohydrate binding proteins and sugar hydrolses / Periplasmic copper-binding protein (NosD) / Parallel beta-helix repeat / Parallel beta-helix repeats / Copper centre Cu(A) / CO II and nitrous oxide reductase dinuclear copper centers signature. / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / Cytochrome C oxidase subunit II, periplasmic domain / Cytochrome c oxidase subunit II-like C-terminal / Cytochrome oxidase subunit II copper A binding domain profile. / Pectin lyase fold / Pectin lyase fold/virulence factor / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / Cupredoxin / 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. / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nitrous-oxide reductase / 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) / Pseudomonas stutzeri (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsZipfel S / Mueller C / Topitsch A / Lutz M / Zhang L / Einsle O
Funding support Germany, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)310656 Germany
German Research Foundation (DFG)CRC1381, 403222702 Germany
German Research Foundation (DFG)RTG2202, 46710898 Germany
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
DepositionNov 18, 2021-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13885.png

Downloads & links

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Map

FileDownload / File: emd_13885.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdensity modified map using Phenix
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.87 Å/pix.
x 240 pix.
= 208.8 Å		0.87 Å/pix.
x 240 pix.
= 208.8 Å		0.87 Å/pix.
x 240 pix.
= 208.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.2765502 - 1.9537505
Average (Standard dev.)-6.6868136e-13 (±0.08129971)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 208.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map after 3D refinement using Relion

Fileemd_13885_additional_1.map
Annotationunsharpened map after 3D refinement using Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heteroheptamer complex of NosZDFY

EntireName: Heteroheptamer complex of NosZDFY
Components
  • Complex: Heteroheptamer complex of NosZDFY
    • 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: Nitrous-oxide reductase
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Heteroheptamer complex of NosZDFY

SupramoleculeName: Heteroheptamer complex of NosZDFY / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: homodimers of NosZ, NosY and NosF; monomer of NosD
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C43(DE3) / Recombinant plasmid: pET

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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 (bacteria)
Molecular weightTheoretical: 48.2585 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (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 (bacteria)
Molecular weightTheoretical: 34.518402 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HHHHHHNAVE IQGVSQRYGS MTVLHDLNLN LGEGEVLGLF GHNGAGKTTS MKLILGLLSP SEGQVKVLGR APNDPQVRRQ LGYLPENVT FYPQLSGRET LRHFARLKGA ALTQVDELLE QVGLAHAADR RVKTYSKGMR QRLGLAQALL GEPRLLLLDQ P TVGLDPIA ...String:
HHHHHHNAVE IQGVSQRYGS MTVLHDLNLN LGEGEVLGLF GHNGAGKTTS MKLILGLLSP SEGQVKVLGR APNDPQVRRQ LGYLPENVT FYPQLSGRET LRHFARLKGA ALTQVDELLE QVGLAHAADR RVKTYSKGMR QRLGLAQALL GEPRLLLLDQ P TVGLDPIA TQDLYLLIDR LRQRGTSIIL CSHVLPGVEA HINRAAILAK GCLQAVGSLS QLRAEAGLPV RIRASGISER DS WLQRWTD AGHSARGLSE SSIEVVAVNG HKLVLLRQLL GEGEPEDIEI HQPSLEDLYR YYMERAGDVR AQEGRL

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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 (bacteria)
Molecular weightTheoretical: 29.449203 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (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: Nitrous-oxide reductase

MacromoleculeName: Nitrous-oxide reductase / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitrous-oxide reductase
Source (natural)Organism: Pseudomonas stutzeri (bacteria)
Molecular weightTheoretical: 71.958555 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSDKDSKNTP QVPEKLGLSR RGFLGASAVT GAAVAATALG GAVMTRESWA QAVKESKQKI HVGPGELDDY YGFWSGGHQG EVRVLGVPS MRELMRIPVF NVDSATGWGL TNESRHIMGD SAKFLNGDCH HPHISMTDGK YDGKYLFIND KANSRVARIR L DIMKCDKM ...String:
MSDKDSKNTP QVPEKLGLSR RGFLGASAVT GAAVAATALG GAVMTRESWA QAVKESKQKI HVGPGELDDY YGFWSGGHQG EVRVLGVPS MRELMRIPVF NVDSATGWGL TNESRHIMGD SAKFLNGDCH HPHISMTDGK YDGKYLFIND KANSRVARIR L DIMKCDKM ITVPNVQAIH GLRLQKVPHT KYVFANAEFI IPHPNDGKVF DLQDENSYTM YNAIDAETME MAFQVIVDGN LD NTDADYT GRFAAATCYN SEKAFDLGGM MRNERDWVVV FDIHAVEAAV KAGDFITLGD SKTPVLDGRK KDGKDSKFTR YVP VPKNPH GCNTSSDGKY FIAAGKLSPT CSMIAIDKLP DLFAGKLADP RDVIVGEPEL GLGPLHTTFD GRGNAYTTLF IDSQ VVKWN MEEAVRAYKG EKVNYIKQKL DVHYQPGHLH ASLCETNEAD GKWLVALSKF SKDRFLPVGP LHPENDQLID ISGDE MKLV HDGPTFAEPH DCIMARRDQI KTKKIWDRND PFFAPTVEMA KKDGINLDTD NKVIRDGNKV RVYMTSMAPA FGVQEF TVK QGDEVTVTIT NIDQIEDVSH GFVVVNHGVS MEISPQQTSS ITFVADKPGL HWYYCSWFCH ALHMEMVGRM MVEPAWS HP QFEK

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 114710

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