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- EMDB-26379: Structure of PA28gamma bound to the human 20S proteasome with C7 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26379
TitleStructure of PA28gamma bound to the human 20S proteasome with C7 symmetry.
Map dataComplex of PA28gamma(REGgamma) bound to the human 20S proteasome with C7 symmetry applied.
Sample
  • Complex: CryoEM structure of the PA28gamma 20S proteasome complex.
    • Protein or peptide: PA28 gamma
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsSmith DM / Thomas T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107129 United States
CitationJournal: J Biol Chem / Year: 2022
Title: Proteasome activator 28γ (PA28γ) allosterically activates trypsin-like proteolysis by binding to the α-ring of the 20S proteasome.
Authors: Taylor A Thomas / David M Smith /
Abstract: Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain ...Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis. Here, by mechanistically decoupling gating effects from active site effects, we unambiguously demonstrate that WT PA28γ allosterically activates the T-L site. We show PA28γ binding increases the Kcat/Km by 13-fold for T-L peptide substrates while having little-to-no effect on hydrolysis kinetics for CT-L or C-L substrates. Furthermore, mutagenesis and domain swaps of PA28γ reveal that it does not select for T-L peptide substrates through either the substrate entry pore or the distal intrinsically disordered region. We also show that a previously reported point mutation can functionally switch PA28γ from a T-L activating to a gate-opening activator in a mutually exclusive fashion. Finally, using cryogenic electron microscopy, we visualized the PA28γ-proteasome complex at 4.3 Å and confirmed its expected quaternary structure. The results of this study provide unambiguous evidence that PA28γ can function by binding the 20S proteasome to allosterically activate the T-L proteolytic site.
History
DepositionMar 5, 2022-
Header (metadata) releaseJul 27, 2022-
Map releaseJul 27, 2022-
UpdateJul 27, 2022-
Current statusJul 27, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26379.png

Downloads & links

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Map

FileDownload / File: emd_26379.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplex of PA28gamma(REGgamma) bound to the human 20S proteasome with C7 symmetry applied.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 512 pix.
= 552.96 Å		1.08 Å/pix.
x 512 pix.
= 552.96 Å		1.08 Å/pix.
x 512 pix.
= 552.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.64
Minimum - Maximum-0.66057116 - 1.4500252
Average (Standard dev.)0.0020204706 (±0.06331693)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 552.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : CryoEM structure of the PA28gamma 20S proteasome complex.

EntireName: CryoEM structure of the PA28gamma 20S proteasome complex.
Components
  • Complex: CryoEM structure of the PA28gamma 20S proteasome complex.
    • Protein or peptide: PA28 gamma

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Supramolecule #1: CryoEM structure of the PA28gamma 20S proteasome complex.

SupramoleculeName: CryoEM structure of the PA28gamma 20S proteasome complex.
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: C7 symmetry is applied.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
Molecular weightTheoretical: 900 KDa

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Macromolecule #1: PA28 gamma

MacromoleculeName: PA28 gamma / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MASLLKVDQE VKLKVDSFRE RITSEAEDLV ANFFPKKLLE LDSFLKEPIL NIHDLTQIHS DMNLPVPDPI LLTNSHDGLD GPTYKKRRLD ECEEAFQGTK VFVMPNGMLK SNQQLVDIIE KVKPEIRLLI EKCNTVKMWV QLLIPRIEDG NNFGVSIQEE TVAELRTVES ...String:
MASLLKVDQE VKLKVDSFRE RITSEAEDLV ANFFPKKLLE LDSFLKEPIL NIHDLTQIHS DMNLPVPDPI LLTNSHDGLD GPTYKKRRLD ECEEAFQGTK VFVMPNGMLK SNQQLVDIIE KVKPEIRLLI EKCNTVKMWV QLLIPRIEDG NNFGVSIQEE TVAELRTVES EAASYLDQIS RYYITRAKLV SKIAKYPHVE DYRRTVTEID EKEYISLRLI ISELRNQYVT LHDMILKNIE KIKRPRSSNA ETLY

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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

Concentration0.2 mg/mL
BufferpH: 7.4 / Component - Concentration: 50.0 mM / Component - Formula: Tris-HCL / Component - Name: Tris buffer
GridModel: Quantifoil / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
DetailsRecombinate human PA28gamma was combine with human 20S proteasome from Hek293 cells. The sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 31712
CTF correctionSoftware - Name: cryoSPARC (ver. 2.3)
Startup modelType of model: NONE / Details: Ab initio (Reference free)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 876
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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