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- EMDB-43290: Cryo-EM structure of Myxococcus xanthus EncA encapsulin shell loa... -

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Basic information

Entry
Database: EMDB / ID: EMD-43290
TitleCryo-EM structure of Myxococcus xanthus EncA encapsulin shell loaded with EncD cargo
Map dataEncA-EncD encapsulin cryo-EM map
Sample
  • Organelle or cellular component: Icosahedral encapsulin EncA particles in complex with cargo protein EncD
    • Protein or peptide: EncA
    • Protein or peptide: Encapsulin nanocompartment cargo protein EncD
KeywordsEncapsulin / EncA / EncD / flavin / flavin mononucleotide / iron / ferroxidase / ferritin / ferric reductase / VIRUS LIKE PARTICLE
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / iron ion transport / intracellular iron ion homeostasis / metal ion binding / Type 1 encapsulin shell protein EncA / Encapsulin nanocompartment cargo protein EncD
Function and homology information
Biological speciesMyxococcus xanthus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsEren E
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)Intramural Research United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity.
Authors: Elif Eren / Norman R Watts / James F Conway / Paul T Wingfield /
Abstract: Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic ...Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe to Fe for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no β-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.
History
DepositionJan 7, 2024-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43290.png

Downloads & links

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Map

FileDownload / File: emd_43290.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEncA-EncD encapsulin cryo-EM map
Voxel sizeX=Y=Z: 0.92 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.04209158 - 0.08621133
Average (Standard dev.)0.0005484793 (±0.0044758148)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 471.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EncA-EncD halfmap1

Fileemd_43290_half_map_1.map
AnnotationEncA-EncD halfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EncA-EncD halfmap2

Fileemd_43290_half_map_2.map
AnnotationEncA-EncD halfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Icosahedral encapsulin EncA particles in complex with cargo prote...

EntireName: Icosahedral encapsulin EncA particles in complex with cargo protein EncD
Components
  • Organelle or cellular component: Icosahedral encapsulin EncA particles in complex with cargo protein EncD
    • Protein or peptide: EncA
    • Protein or peptide: Encapsulin nanocompartment cargo protein EncD

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Supramolecule #1: Icosahedral encapsulin EncA particles in complex with cargo prote...

SupramoleculeName: Icosahedral encapsulin EncA particles in complex with cargo protein EncD
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Myxococcus xanthus (bacteria)

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Macromolecule #1: EncA

MacromoleculeName: EncA / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Myxococcus xanthus (bacteria)
Molecular weightTheoretical: 33.505074 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHMPL EPHFMPDFLG HAENPLREEE WARLNETVIQ VARRSLVGRR ILDIYGPLGA GVQTVPYDEF QGVSPGAVDI VGEQETAMV FTDARKFKTI PIIYKDFLLH WRDIEAARTH NMPLDVSAAA GAAALCAQQE DELIFYGDAR LGYEGLMTAN G RLTVPLGD ...String:
MHHHHHHMPL EPHFMPDFLG HAENPLREEE WARLNETVIQ VARRSLVGRR ILDIYGPLGA GVQTVPYDEF QGVSPGAVDI VGEQETAMV FTDARKFKTI PIIYKDFLLH WRDIEAARTH NMPLDVSAAA GAAALCAQQE DELIFYGDAR LGYEGLMTAN G RLTVPLGD WTSPGGGFQA IVEATRKLNE QGHFGPYAVV LSPRLYSQLH RIYEKTGVLE IETIRQLASD GVYQSNRLRG ES GVVVSTG RENMDLAVSM DMVAAYLGAS RMNHPFRVLE ALLLRIKHPD AICTLEGAGA TERR

UniProtKB: Type 1 encapsulin shell protein EncA

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Macromolecule #2: Encapsulin nanocompartment cargo protein EncD

MacromoleculeName: Encapsulin nanocompartment cargo protein EncD / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Myxococcus xanthus (bacteria)
Molecular weightTheoretical: 859.992 Da
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GLTVGSLRG

UniProtKB: Encapsulin nanocompartment cargo protein EncD

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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

Concentration0.5 mg/mL
BufferpH: 7.3 / Details: 20mM HEPES, 150mM NaCl
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7s4q

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5) / Number images used: 47437
FSC plot (resolution estimation)

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