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- EMDB-45001: Structure of Mnx H340A complex from Bacillus sp. PL-12 -

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Basic information

Entry
Database: EMDB / ID: EMD-45001
TitleStructure of Mnx H340A complex from Bacillus sp. PL-12
Map data
Sample
  • Complex: Mnx H340A complex
    • Protein or peptide: MnxG
    • Protein or peptide: MnxE
    • Protein or peptide: MnxF
KeywordsComplex / multicopper oxidase / manganese biomineralization / METAL TRANSPORT
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. (in: firmicutes) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsNovikova IV / Evans JE
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45001.png

Downloads & links

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Map

FileDownload / File: emd_45001.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 330 pix.
= 224.4 Å		0.68 Å/pix.
x 330 pix.
= 224.4 Å		0.68 Å/pix.
x 330 pix.
= 224.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.68 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.071
Minimum - Maximum-0.08595846 - 0.21652254
Average (Standard dev.)0.0013756333 (±0.011571339)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 224.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45001_msk_1.map
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Half map: #2

Fileemd_45001_half_map_1.map
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Half map: #1

Fileemd_45001_half_map_2.map
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Sample components

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Entire : Mnx H340A complex

EntireName: Mnx H340A complex
Components
  • Complex: Mnx H340A complex
    • Protein or peptide: MnxG
    • Protein or peptide: MnxE
    • Protein or peptide: MnxF

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Supramolecule #1: Mnx H340A complex

SupramoleculeName: Mnx H340A complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus sp. (in: firmicutes) (bacteria)
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: MnxG

MacromoleculeName: MnxG / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus sp. (in: firmicutes) (bacteria)
Molecular weightTheoretical: 138.340672 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MLRKFHVVGI STRIVVNTFG DHNPNGRIYV LKENESKLKD LVRKNPYKPI DLVQPLAIRA NEGDIVEILF ENQLSFSAGM HFQEADYSV LSSDGADAGY NPDTTVEPGG EILYRLNVNQ EGICFFTDLG NVSSTEQGSS VQGLFGALLV QKRGSSWTDP V TGGPINSG ...String:
MLRKFHVVGI STRIVVNTFG DHNPNGRIYV LKENESKLKD LVRKNPYKPI DLVQPLAIRA NEGDIVEILF ENQLSFSAGM HFQEADYSV LSSDGADAGY NPDTTVEPGG EILYRLNVNQ EGICFFTDLG NVSSTEQGSS VQGLFGALLV QKRGSSWTDP V TGGPINSG VYADIHHPFL PSFREYAWFF NDEMEIRDLT GERPLNPMTN QEAESFHGVN LRYEPMTNRK RLMEAGVVCP DC DSEEVHH DSWVFGDPAT PILRGYVGDP AVIRLIHGGV KETHVFHYHV HQWLGDSSNI NAEILDAQSI SPQTHYSIQP LYG LGSLHG AIGDSIIHCH LYPAFGIGMW GMNRVFDTLQ DGSQCYPNGV RIKALMPLPD RPEPPKPTPE KPGFPNFIPG KVGY KAPRP PLGIVGGREM TELERNAAIE NPRPGAVFVD PCLDQDPVVV EFNVSAIEMP VVYNKQGWHD PKARFYVMDE DLDDI LSGK KEPEPLVFHV PAGTCIRMNY TNRMPHILDG DAFQLVTRTY ENGFHIHFVK FDVLACDGGN VGWNYDSAVL PGQTIR YEW YAETELKAFF FHDHLFANSH QQHGVFGAGV IQPRFSKFLD SRTGDEVDHG TQISVEHPLI PDYRDQTLFV HDFALLF DK NGRPIQPPEY PGSEDDPGVF GVNFKCEPLK FRLGEDCDPA YSFSSYVHGD PVTPILRAYE GDPIRIRLLQ GAHEESHS F NIHGLRWKEE RPDLGSSMKA QQHIGISESF TFETEIPASG DYLWAFEDEE DVWLGTWGLI RAYKGRMEDL IVLTDREAL PEGSAETPKP TGKPPEKANP LASLPPGAYQ GSPVKKFEVV AFQTPIQYNS YGDHDPYGII FALKEDVEDI LTGKKNPVPL ILRANVGDL VEVTLTSELK KELFPFQDGI HPYPPVKEQS FYPPSLRISL HTSLLNYDVK TSSGDTVGYN PDQTVGPGET I TYRWFVDG QFGMCSMWDM ADLRNHRSFG TFGAFVAESR FTTYLDPYSL EKAITGENVI LRHPLLPATR EFVLILHDGV RL EDKDGKV IIDPMDGVVP DTEELEEVDT YDYGSRGFNY RSERLINRYK EHPVMHELFS SEVFGDPATP LFEAYPGEPV VMR ITTPAE RRRAHTFHLH GHYWKFDSKD LDSRIQSFLG HMVTGHTDDL RLIGGAGGVF NFPGDYLYRS GNIRWDIELG MWGI FRVHK DSKENLPRLE EVEGGWDNEE KA

UniProtKB: MnxG

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Macromolecule #2: MnxE

MacromoleculeName: MnxE / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Bacillus sp. (in: firmicutes) (bacteria)
Molecular weightTheoretical: 12.185235 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MHDSPLKSLS AASNVASVND PLFDFFNKHM GKQILIITES SQLNILGQTF RPIFCGKVAE VEPGHLTLSP VTIKILNAPF HKFPIPLSI PFEKIAHFTT DVDCSMRIPL V

UniProtKB: MnxE

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Macromolecule #3: MnxF

MacromoleculeName: MnxF / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Bacillus sp. (in: firmicutes) (bacteria)
Molecular weightTheoretical: 12.026734 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MEALFPMSTD YSKMTDVNEI HDSAILEHFR NGIGHKTLVI SPSYPYMFVG IIKELIGDTV MIDVETTHFA QLENREWYIH IHNIEVFYI ERPGAPKIPK LEDY

UniProtKB: MnxF

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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

Concentration4.5 mg/mL
BufferpH: 7.8
VitrificationCryogen name: ETHANE / Chamber humidity: 90 %
Detailscontains 80mM CHAPSO

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / 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 / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 98663
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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