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- PDB-7o14: ABC transporter NosDFY, nucleotide-free in lipid nanodisc, R-domain 1 -

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Basic information

Entry
Database: PDB / ID: 7o14
TitleABC transporter NosDFY, nucleotide-free in lipid nanodisc, R-domain 1
Components
  • Probable ABC transporter ATP-binding protein NosF
  • Probable ABC transporter binding protein NosD
  • Probable ABC transporter permease protein NosY
KeywordsMEMBRANE PROTEIN / ABC Transporter complex / nucleotide-free
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 / Parallel beta-helix repeats ...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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM
AuthorsMueller, C. / Zhang, L. / Lu, W. / Einsle, O. / Du, J.
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Probable ABC transporter ATP-binding protein NosF
C: Probable ABC transporter ATP-binding protein NosF
D: Probable ABC transporter permease protein NosY
E: Probable ABC transporter permease protein NosY
A: Probable ABC transporter binding protein NosD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)174,8256
Polymers174,8005
Non-polymers241
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13570 Å2
ΔGint-76 kcal/mol
Surface area60620 Å2

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Components

#1: Protein Probable ABC transporter ATP-binding protein NosF


Mass: 33821.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P19844
#2: Protein Probable ABC transporter permease protein NosY


Mass: 29449.203 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P19845
#3: Protein Probable ABC transporter binding protein NosD


Mass: 48258.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P19843
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ABC Transporter NosFY in complex with accessory periplasmic protein NosD
Type: COMPLEX
Details: Nucleotide-free state in lipid nanodisc, R-domain conformation 1
Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Pseudomonas stutzeri ATCC 14405 = CCUG 16156 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingAverage exposure time: 1 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k)
Image scansMovie frames/image: 40

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
9GctfCTF correction
1Gautomatchparticle selection
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionNum. of particles: 130946 / Symmetry type: POINT
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008812009
ELECTRON MICROSCOPYf_angle_d1.150916302
ELECTRON MICROSCOPYf_chiral_restr0.07041880
ELECTRON MICROSCOPYf_plane_restr0.00742092
ELECTRON MICROSCOPYf_dihedral_angle_d12.24537109

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