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- EMDB-16655: Low-resolution cryo-EM structure of the NorQD chaperone complex f... -

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Basic information

Entry
Database: EMDB / ID: EMD-16655
TitleLow-resolution cryo-EM structure of the NorQD chaperone complex from Paracoccus denitrificans
Map dataStructure of the NorQD complex from Paracoccus denitrificans
Sample
  • Complex: NorQD complex
    • Protein or peptide: Pd. NorQ
    • Protein or peptide: Pd. NorD
KeywordsMoxR AAA+ and VWA domain proteins / CHAPERONE
Biological speciesParacoccus denitrificans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.09 Å
AuthorsAdelroth P / Carroni M / Kahle M / Appelgren S / Elofsson A
Funding support Sweden, 3 items
OrganizationGrant numberCountry
Swedish Research CouncilVR- 2015-04512 and 2019-04124 Sweden
Swedish Research CouncilVR-2016-06301 Sweden
Knut and Alice Wallenberg Foundation31001512 Sweden
CitationJournal: BMC Biol / Year: 2023
Title: Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins.
Authors: Maximilian Kahle / Sofia Appelgren / Arne Elofsson / Marta Carroni / Pia Ädelroth /
Abstract: BACKGROUND: NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (Fe) cofactor insertion into ...BACKGROUND: NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (Fe) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed Fe insertion, and the general mechanism of the MoxR/VWA interacting protein families.
RESULTS: We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low- ...RESULTS: We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that "plugs" the NorQ ring pore to the VWA domain of NorD with a protruding "finger" inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.
CONCLUSIONS: Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.
History
DepositionFeb 6, 2023-
Header (metadata) releaseFeb 22, 2023-
Map releaseFeb 22, 2023-
UpdateSep 27, 2023-
Current statusSep 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16655.png

Downloads & links

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Map

FileDownload / File: emd_16655.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the NorQD complex from Paracoccus denitrificans
Voxel sizeX=Y=Z: 0.8464 Å
Density
Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.044013973 - 0.1488081
Average (Standard dev.)0.0012516181 (±0.011667542)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 216.6784 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Structure of the NorQD complex from Paracoccus denitrificans halfA

Fileemd_16655_half_map_1.map
AnnotationStructure of the NorQD complex from Paracoccus denitrificans halfA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of the NorQD complex from Paracoccus denitrificans halfB

Fileemd_16655_half_map_2.map
AnnotationStructure of the NorQD complex from Paracoccus denitrificans halfB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NorQD complex

EntireName: NorQD complex
Components
  • Complex: NorQD complex
    • Protein or peptide: Pd. NorQ
    • Protein or peptide: Pd. NorD

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Supramolecule #1: NorQD complex

SupramoleculeName: NorQD complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Paracoccus denitrificans (bacteria)

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Macromolecule #1: Pd. NorQ

MacromoleculeName: Pd. NorQ / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Paracoccus denitrificans (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASNAHVKTQ GNGAVDAPFY LPQGDEVAVF EAAAANDLPV LLKGPTGCGK TRFVAHMAAR LG RPLYTVA CHDDLSAADL IGRYLLKGGE TVWTDGPLTR AVREGAICYL DEVVEARKDV TVV LHPLTD DRRILPIDRT GEEIEAAPGF MLVASYNPGY QNILKTLKPS ...String:
MASNAHVKTQ GNGAVDAPFY LPQGDEVAVF EAAAANDLPV LLKGPTGCGK TRFVAHMAAR LG RPLYTVA CHDDLSAADL IGRYLLKGGE TVWTDGPLTR AVREGAICYL DEVVEARKDV TVV LHPLTD DRRILPIDRT GEEIEAAPGF MLVASYNPGY QNILKTLKPS TRQRFVAMEF DFPE PAREV EIVARESGLD RDRTLGLVRL AGKIRGLKGQ DLEEGVSTRL VVYAASLTRR GMNLD RAIE AAMIEPLTDD AEVKRGLRDL AAAIFG

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Macromolecule #2: Pd. NorD

MacromoleculeName: Pd. NorD / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Paracoccus denitrificans (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHSRG LDLEPWEPEE TVGKIWHVW ASSFGAPQAF E DQAVALSE VSGRLAVLFR AL GGGAAVE IRPAAVQASH HRI GWLRRL GTPAETVPHA SFDG EILRL PERLSVLPSR QANGA LFLW LAACAAHGSL APAQGD PLC RDLVRLGAAQ RAVEATL QD ...String:
MHHHHHHSRG LDLEPWEPEE TVGKIWHVW ASSFGAPQAF E DQAVALSE VSGRLAVLFR AL GGGAAVE IRPAAVQASH HRI GWLRRL GTPAETVPHA SFDG EILRL PERLSVLPSR QANGA LFLW LAACAAHGSL APAQGD PLC RDLVRLGAAQ RAVEATL QD APGLTGLYDD LAELVLSL R PVAPLPPAEA VVEALARHL LGDPAPLPPL ARDWLAMLDD PQVKAPRDY RPMRPVPLWP D LALPETTL AAAPGDAPDG IA ADPANAR MFRARRRQSD QPQ RRDSLI LHKFEALLSW ADLM NLNRH VDDDDQDDAK KAAED QEEL GLGQVSKAPA TRLRLH LDL APEDADLEAV AGIRTYP EW DARRGRYLAH HVRVLENR A PEHDEALTPD PRAQTRIRA VRRQFEALRP GRLITTGHRD GDELDAELT VRAAADLRAT G QGSDRIWR QSRPLARNLA VS ILLDVSR STESAVTGRA VIE IEREAL AALAWGLDAC GDRF AINAF SSLKRDRVFL SACKD FDEP MGAAIERRIA GLRPRF YTR LGAGIRHASA GLSAQAS SR RLLLVITDGK PNDLDHYE G RHGIEDSAMA VREARRAGH AVHGITVDRD AKSWFPRIFG QGGFSLIPH PDRLLAALPV I YRQLVA

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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

Concentration1 mg/mL
BufferpH: 8 / Details: 50mM TRIS/HCl, 150 mM NaCl, 10% glycerol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.09 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 507400
Initial angle assignmentType: OTHER / Details: SGD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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