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- PDB-9bxa: Structure of Mnx H340A complex from Bacillus sp. PL-12 -

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Basic information

Entry
Database: PDB / ID: 9bxa
TitleStructure of Mnx H340A complex from Bacillus sp. PL-12
Components
  • MnxE
  • MnxF
  • MnxG
KeywordsMETAL TRANSPORT / Complex / multicopper oxidase / manganese biomineralization
Function / homologyMulticopper oxidase, copper-binding site / Multicopper oxidases signature 2. / Multicopper oxidase, N-terminal / Multicopper oxidase / Cupredoxin / copper ion binding / MnxE / MnxF / MnxG
Function and homology information
Biological speciesBacillus sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsNovikova, I.V. / Evans, J.E.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)50773 (under EMSL) United States
CitationJournal: J Am Chem Soc / Year: 2024
Title: Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.
Authors: Irina V Novikova / Alexandra V Soldatova / Trevor H Moser / Stephanie M Thibert / Christine A Romano / Mowei Zhou / Bradley M Tebo / James E Evans / Thomas G Spiro /
Abstract: The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O directly, via ...The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxEF cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.
History
DepositionMay 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.2Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: MnxG
B: MnxE
C: MnxE
D: MnxE
E: MnxF
F: MnxF
G: MnxF


Theoretical massNumber of molelcules
Total (without water)210,9777
Polymers210,9777
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein MnxG


Mass: 138340.672 Da / Num. of mol.: 1 / Mutation: H340A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. (in: firmicutes) (bacteria)
Gene: mnxG
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A7KBU7
#2: Protein MnxE


Mass: 12185.235 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. (in: firmicutes) (bacteria)
Gene: mnxE
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A7KBU5
#3: Protein MnxF


Mass: 12026.734 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus sp. (in: firmicutes) (bacteria)
Gene: mnxF
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A7KBU6
Has protein modificationY

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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: Mnx H340A complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: YES
Source (natural)Organism: Bacillus sp. (in: firmicutes) (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.8
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: contains 80mM CHAPSO
VitrificationCryogen name: ETHANE / Humidity: 90 %

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98663 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 187.03 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002114464
ELECTRON MICROSCOPYf_angle_d0.538519678
ELECTRON MICROSCOPYf_chiral_restr0.04622101
ELECTRON MICROSCOPYf_plane_restr0.00422592
ELECTRON MICROSCOPYf_dihedral_angle_d4.04931900

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