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- PDB-5lzp: Binding of the C-terminal GQYL motif of the bacterial proteasome ... -

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Basic information

Entry
Database: PDB / ID: 5lzp
TitleBinding of the C-terminal GQYL motif of the bacterial proteasome activator Bpa to the 20S proteasome
Components
  • Bacterial proteasome activatorProteasome accessory factor E
  • Proteasome subunit alpha
  • Proteasome subunit beta
KeywordsHYDROLASE / Proteasome / Proteasome activator / protein degradation / complex
Function / homology
Function and homology information


symbiont-mediated perturbation of host defenses / proteasome accessory complex / positive regulation of proteasomal protein catabolic process / zymogen binding / proteasome binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / peptidoglycan-based cell wall ...symbiont-mediated perturbation of host defenses / proteasome accessory complex / positive regulation of proteasomal protein catabolic process / zymogen binding / proteasome binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / peptidoglycan-based cell wall / proteolysis involved in protein catabolic process / proteasomal protein catabolic process / modification-dependent protein catabolic process / protein homooligomerization / extracellular region / plasma membrane / cytoplasm
Similarity search - Function
Bacterial proteasome activator / Bacterial proteasome activator / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. ...Bacterial proteasome activator / Bacterial proteasome activator / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Proteasome subunit beta / Proteasome subunit alpha / Bacterial proteasome activator
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBolten, M. / Delley, C.L. / Leibundgut, M. / Boehringer, D. / Ban, N. / Weber-Ban, E.
CitationJournal: Structure / Year: 2016
Title: Structural Analysis of the Bacterial Proteasome Activator Bpa in Complex with the 20S Proteasome.
Authors: Marcel Bolten / Cyrille L Delley / Marc Leibundgut / Daniel Boehringer / Nenad Ban / Eilika Weber-Ban /
Abstract: Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial ...Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex.
History
DepositionSep 30, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 23, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2016Group: Database references
Revision 1.2Jul 26, 2017Group: Experimental preparation / Category: em_sample_support
Item: _em_sample_support.details / _em_sample_support.grid_type
Revision 1.3Aug 2, 2017Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Oct 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.5Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
0: Proteasome subunit alpha
1: Bacterial proteasome activator
2: Proteasome subunit alpha
3: Bacterial proteasome activator
4: Proteasome subunit alpha
5: Bacterial proteasome activator
6: Proteasome subunit alpha
7: Bacterial proteasome activator
8: Proteasome subunit alpha
A: Proteasome subunit beta
B: Proteasome subunit alpha
C: Proteasome subunit beta
D: Proteasome subunit alpha
E: Proteasome subunit beta
F: Proteasome subunit beta
G: Proteasome subunit beta
H: Proteasome subunit alpha
I: Proteasome subunit beta
J: Proteasome subunit alpha
K: Proteasome subunit beta
L: Proteasome subunit beta
M: Proteasome subunit alpha
N: Proteasome subunit beta
O: Proteasome subunit beta
P: Proteasome subunit beta
Q: Proteasome subunit alpha
R: Proteasome subunit beta
S: Proteasome subunit alpha
T: Proteasome subunit beta
U: Proteasome subunit beta
V: Bacterial proteasome activator
W: Proteasome subunit alpha
X: Bacterial proteasome activator
Y: Proteasome subunit alpha
Z: Bacterial proteasome activator


Theoretical massNumber of molelcules
Total (without water)859,29935
Polymers859,29935
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area75430 Å2
ΔGint-59 kcal/mol
Surface area233980 Å2
MethodPISA

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Components

#1: Protein
Proteasome subunit alpha / / 20S proteasome alpha subunit / Proteasome core protein PrcA


Mass: 26024.971 Da / Num. of mol.: 14 / Mutation: M1_I7del
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: prcA, Rv2109c / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P9WHU1, proteasome endopeptidase complex
#2: Protein
Bacterial proteasome activator / Proteasome accessory factor E


Mass: 19792.113 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: bpa, Rv3780, MTCY13D12.14 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WKX3
#3: Protein
Proteasome subunit beta / / 20S proteasome beta subunit / Proteasome core protein PrcB


Mass: 25457.504 Da / Num. of mol.: 14 / Mutation: T1A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: prcB, Rv2110c / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P9WHT9, proteasome endopeptidase complex

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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: proteasome in complex with bacterial proteasome activator
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.93 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria) / Cellular location: cytoplasm
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.102 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil R 2/2 with an additional thin carbon layer
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 280.5 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 FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 100000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1
Details: Drift corrected in post-processing. 4 images per hole.
Image scansSampling size: 14 µm / Movie frames/image: 7

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Processing

SoftwareName: PHENIX / Version: (1.10_2155: ???) / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN1.9particle selectionbatchboxer
4CTFFIND4.0.17CTF correction
7UCSF Chimera1.10.2model fitting
9PHENIX1.10-2155model refinement
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
12RELION1.4classification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48799 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementResolution: 3.45→403.2 Å / SU ML: 0.73 / σ(F): 1.99 / Phase error: 34.64 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.3144 99751 2.99 %
Rwork0.3149 --
obs0.3149 3340039 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00747355
ELECTRON MICROSCOPYf_angle_d0.88464086
ELECTRON MICROSCOPYf_dihedral_angle_d13.32628475
ELECTRON MICROSCOPYf_chiral_restr0.0497268
ELECTRON MICROSCOPYf_plane_restr0.0068464

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