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- EMDB-44914: Thermoplasma acidophilum 20S proteasome alphaV24F -

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Basic information

Entry
Database: EMDB / ID: EMD-44914
TitleThermoplasma acidophilum 20S proteasome alphaV24F
Map data
Sample
  • Complex: Thermoplasma acidophilum 20S proteasome alphaV24F
KeywordsProtease / threonine protease / endopeptidase activity / HYDROLASE
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsChuah J / Smith D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01AG064188 United States
CitationJournal: bioRxiv / Year: 2025
Title: Occupancy of the HbYX hydrophobic pocket is sufficient to induce gate opening in the archaeal 20S proteasomes.
Authors: Janelle J Y Chuah / Madalena R Daugherty / David M Smith /
Abstract: Enhancing proteasome function has been a long-standing but challenging target of interest for the potential treatment of neurodegenerative diseases, emphasizing the importance of understanding ...Enhancing proteasome function has been a long-standing but challenging target of interest for the potential treatment of neurodegenerative diseases, emphasizing the importance of understanding proteasome activation mechanisms. Most proteasome activator complexes use the C-terminal HbYX (hydrophobic-tyrosine-almost any residue) motif to bind and trigger gate-opening in the 20S proteasome. This study defines a critical molecular interaction in the HbYX mechanism that triggers gate opening. We focus on the Hb site interaction and find it plays a surprisingly central and crucial role in driving the allosteric conformational changes that induce gate opening in the archaeal 20S. We examined the cryo-EM structure of two mutant archaeal proteasomes, αV24Y T20S and αV24F T20S. These two mutants were engineered to place a bulky aromatic residue in the HbYX hydrophobic pocket; both mutants are highly active, though their mechanisms of activation are undefined. Collectively, our findings indicate that the interaction between the Hb group of the HbYX motif and its corresponding hydrophobic pocket is sufficient to induce gate opening in a mechanistically similar way to the HbYX motif. The activation mechanism studied here involves the expansion of the hydrophobic binding site, allosterically altering the state of the IT switch thus triggering gate-opening. Furthermore, we show that the canonical αK66 residue, previously understood to be critical for proteasome activator binding, also plays a key role in stabilizing the open gate, irrespective of activator binding. This study differentiates between the residues in the HbYX motif that support binding interactions ("YX") versus those that allosterically contribute to gate opening ("Hb"). The insights reported here will guide future drug development efforts, particularly in designing small molecule proteasome activators, by targeting the identified hydrophobic pocket.
History
DepositionMay 17, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44914.png

Downloads & links

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Map

FileDownload / File: emd_44914.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.54 Å/pix.
x 512 pix.
= 276.48 Å		0.54 Å/pix.
x 512 pix.
= 276.48 Å		0.54 Å/pix.
x 512 pix.
= 276.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.54 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0473
Minimum - Maximum-0.09090464 - 0.21921718
Average (Standard dev.)0.00072851044 (±0.010095118)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44914_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_44914_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Thermoplasma acidophilum 20S proteasome alphaV24F

EntireName: Thermoplasma acidophilum 20S proteasome alphaV24F
Components
  • Complex: Thermoplasma acidophilum 20S proteasome alphaV24F

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Supramolecule #1: Thermoplasma acidophilum 20S proteasome alphaV24F

SupramoleculeName: Thermoplasma acidophilum 20S proteasome alphaV24F / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 700 kDa/nm

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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

Concentration1 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: SUPER-RESOLUTION / 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 / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 542722
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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