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- PDB-8vjn: Myxococcus xanthus encapsulin cargo protein EncD in complex with ... -

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Entry
Database: PDB / ID: 8vjn
TitleMyxococcus xanthus encapsulin cargo protein EncD in complex with flavin mononucleotide
ComponentsEncapsulin nanocompartment cargo protein EncD
KeywordsFLAVOPROTEIN / Encapsulin / cargo protein / EncD / flavin / flavin mononucleotide / EncA
Function / homologyencapsulin nanocompartment / intracellular iron ion homeostasis / metal ion binding / FLAVIN MONONUCLEOTIDE / Encapsulin nanocompartment cargo protein EncD
Function and homology information
Biological speciesMyxococcus xanthus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.31 Å
AuthorsEren, E.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: 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.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Encapsulin nanocompartment cargo protein EncD
B: Encapsulin nanocompartment cargo protein EncD
C: Encapsulin nanocompartment cargo protein EncD
D: Encapsulin nanocompartment cargo protein EncD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,4796
Polymers30,5674
Non-polymers9132
Water1,53185
1
A: Encapsulin nanocompartment cargo protein EncD
C: Encapsulin nanocompartment cargo protein EncD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,7403
Polymers15,2832
Non-polymers4561
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2940 Å2
ΔGint-18 kcal/mol
Surface area6020 Å2
MethodPISA
2
B: Encapsulin nanocompartment cargo protein EncD
D: Encapsulin nanocompartment cargo protein EncD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,7403
Polymers15,2832
Non-polymers4561
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2920 Å2
ΔGint-18 kcal/mol
Surface area6180 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.566, 27.999, 86.469
Angle α, β, γ (deg.)90.00, 96.83, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
Encapsulin nanocompartment cargo protein EncD


Mass: 7641.625 Da / Num. of mol.: 4 / Fragment: residues 1-59
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Myxococcus xanthus (bacteria) / Gene: encD, MXAN_2410 / Production host: Escherichia coli (E. coli) / References: UniProt: Q1D9P3
#2: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 85 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 1.72 Å3/Da / Density % sol: 28.56 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, sitting drop / pH: 5
Details: 25% PEG 1500, 0.1 M PCTP (sodium propionate, sodium cacodylate and Bis-Tris propane, 2:1:2)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.9774 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 23, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9774 Å / Relative weight: 1
ReflectionResolution: 2.31→42.93 Å / Num. obs: 9203 / % possible obs: 96.94 % / Redundancy: 2 % / CC1/2: 0.997 / Rmerge(I) obs: 0.058 / Net I/σ(I): 8.93
Reflection shellResolution: 2.31→2.4 Å / Redundancy: 2 % / Rmerge(I) obs: 0.84 / Num. unique obs: 726 / CC1/2: 0.37 / % possible all: 80

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Processing

Software
NameVersionClassification
PHENIX1.18.2-3874refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.31→32.54 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.91 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2306 919 10 %Random selection
Rwork0.1828 ---
obs0.1879 9189 96.98 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.31→32.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1659 0 62 85 1806
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009
X-RAY DIFFRACTIONf_angle_d1.052
X-RAY DIFFRACTIONf_dihedral_angle_d16.803246
X-RAY DIFFRACTIONf_chiral_restr0.052250
X-RAY DIFFRACTIONf_plane_restr0.012302
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.31-2.430.31021080.2561988X-RAY DIFFRACTION84
2.43-2.580.34641290.24181154X-RAY DIFFRACTION96
2.58-2.780.32191330.23561199X-RAY DIFFRACTION100
2.78-3.060.27411350.20611220X-RAY DIFFRACTION100
3.06-3.510.22421350.18891214X-RAY DIFFRACTION100
3.51-4.420.19061360.14341218X-RAY DIFFRACTION100
4.42-32.540.19571430.16921277X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: 22.0562 Å / Origin y: -5.1802 Å / Origin z: 21.4996 Å
111213212223313233
T0.2599 Å2-0.0056 Å2-0.0052 Å2-0.3045 Å20.0271 Å2--0.2785 Å2
L-0.4094 °20.0877 °2-0.3456 °2-0.4191 °2-0.1964 °2--0.4534 °2
S0.0234 Å °-0.0145 Å °0.0037 Å °-0.0096 Å °0.0019 Å °-0.0045 Å °-0.0047 Å °0.0086 Å °-0 Å °
Refinement TLS groupSelection details: all

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