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- PDB-9d5a: Structure of Citrobacter multi-ubiquitin protein, local refinemen... -

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Basic information

Entry
Database: PDB / ID: 9d5a
TitleStructure of Citrobacter multi-ubiquitin protein, local refinement of one full-length protomer
ComponentsMulti-ubiquitin domain-containing protein
KeywordsPROTEIN BINDING / filament / beta-grasp
Biological speciesCitrobacter sp. RHBSTW-00271 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsGong, M. / Gu, Y. / Corbett, K.D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM144121 United States
CitationJournal: Structure / Year: 2025
Title: Structural diversity and oligomerization of bacterial ubiquitin-like proteins.
Authors: Minheng Gong / Qiaozhen Ye / Yajie Gu / Lydia R Chambers / Andrey A Bobkov / Neal K Arakawa / Mariusz Matyszewski / Kevin D Corbett /
Abstract: Bacteria possess a variety of operons with homology to eukaryotic ubiquitination pathways that encode predicted E1, E2, E3, deubiquitinase, and ubiquitin-like proteins. Some of these pathways have ...Bacteria possess a variety of operons with homology to eukaryotic ubiquitination pathways that encode predicted E1, E2, E3, deubiquitinase, and ubiquitin-like proteins. Some of these pathways have recently been shown to function in anti-bacteriophage immunity, but the biological functions of others remain unknown. Here, we show that ubiquitin-like proteins in two bacterial operon families show surprising architectural diversity, possessing one to three β-grasp domains preceded by diverse N-terminal domains. We find that a large group of bacterial ubiquitin-like proteins possess three β-grasp domains and form homodimers and helical filaments mediated by conserved Ca ion binding sites. Our findings highlight a distinctive mode of self-assembly for ubiquitin-like proteins and suggest that Ca-mediated ubiquitin-like protein filament assembly and/or disassembly enables cells to sense and respond to stress conditions that alter intracellular metal ion concentration.
History
DepositionAug 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Apr 2, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Apr 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Multi-ubiquitin domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,5776
Polymers28,3771
Non-polymers2005
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Multi-ubiquitin domain-containing protein


Mass: 28376.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Citrobacter sp. RHBSTW-00271 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Filament assembly of Citrobacter sp. ubiquitin-like (Ubl) protein with Ca ion
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 20 kDa/nm / Experimental value: NO
Source (natural)Organism: Citrobacter sp. RHBSTW-00271 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisC4H11NO31
2300 mMsodium chlorideNaCl1
35 mMCalciumMgCl21
41 mMDTTC4H10O2S21
55 percentglycerolC3H8O31
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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: 2200 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k 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: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 148697 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 124.87 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00421729
ELECTRON MICROSCOPYf_angle_d0.41832342
ELECTRON MICROSCOPYf_chiral_restr0.048265
ELECTRON MICROSCOPYf_plane_restr0.0037310
ELECTRON MICROSCOPYf_dihedral_angle_d9.0286661

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