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- EMDB-28876: Thermoplasma acidophilum 20S proteasome - L81Y mutation in alpha ... -

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Basic information

Entry
Database: EMDB / ID: EMD-28876
TitleThermoplasma acidophilum 20S proteasome - L81Y mutation in alpha subunit
Map dataL81Y T20S EM Map
Sample
  • Complex: Thermoplasma acidophilum 20S proteasome - alphaL81Y mutant
    • Protein or peptide: Proteasome subunit beta
    • Protein or peptide: Proteasome subunit alpha
KeywordsProtease / threonine protease / endopeptidase activity / HYDROLASE
Function / homology
Function and homology information


proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / cytoplasm
Similarity search - Function
Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome B-type subunit ...Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Proteasome subunit alpha / Proteasome subunit beta
Similarity search - Component
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.28 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: Commun Biol / Year: 2023
Title: High resolution structures define divergent and convergent mechanisms of archaeal proteasome activation.
Authors: Janelle J Y Chuah / Matthew S Rexroad / David M Smith /
Abstract: Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it's crucial to understand ...Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it's crucial to understand proteasome activation mechanisms. A hydrophobic-tyrosine-any residue (HbYX) motif on the C-termini of proteasome-activating complexes independently triggers gate-opening of the 20S core particle for protein degradation; however, the causal allosteric mechanism remains unclear. Our study employs a structurally irreducible dipeptide HbYX mimetic to investigate the allosteric mechanism of gate-opening in the archaeal proteasome. High-resolution cryo-EM structures pinpoint vital residues and conformational changes in the proteasome α-subunit implicated in HbYX-dependent activation. Using point mutations, we simulated the HbYX-bound state, providing support for our mechanistic model. We discerned four main mechanistic elements triggering gate-opening: 1) back-loop rearrangement adjacent to K66, 2) intra- and inter- α subunit conformational changes, 3) occupancy of the hydrophobic pocket, and 4) a highly conserved isoleucine-threonine pair in the 20S channel stabilizing the open and closed states, termed the "IT switch." Comparison of different complexes unveiled convergent and divergent mechanism of 20S gate-opening among HbYX-dependent and independent activators. This study delivers a detailed molecular model for HbYX-dependent 20S gate-opening, enabling the development of small molecule proteasome activators that hold promise to treat neurodegenerative diseases.
History
DepositionNov 16, 2022-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28876.png

Downloads & links

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Map

FileDownload / File: emd_28876.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationL81Y T20S EM Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.63 Å/pix.
x 512 pix.
= 323.999 Å		0.63 Å/pix.
x 512 pix.
= 323.999 Å		0.63 Å/pix.
x 512 pix.
= 323.999 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.63281 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0614
Minimum - Maximum-0.11549361 - 0.33569553
Average (Standard dev.)0.0004697431 (±0.01507264)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 323.99872 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_28876_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: L81Y T20S EM Map Half A

Fileemd_28876_half_map_1.map
AnnotationL81Y T20S EM Map Half A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: L81Y T20S EM Map Half B

Fileemd_28876_half_map_2.map
AnnotationL81Y T20S EM Map Half B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thermoplasma acidophilum 20S proteasome - alphaL81Y mutant

EntireName: Thermoplasma acidophilum 20S proteasome - alphaL81Y mutant
Components
  • Complex: Thermoplasma acidophilum 20S proteasome - alphaL81Y mutant
    • Protein or peptide: Proteasome subunit beta
    • Protein or peptide: Proteasome subunit alpha

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Supramolecule #1: Thermoplasma acidophilum 20S proteasome - alphaL81Y mutant

SupramoleculeName: Thermoplasma acidophilum 20S proteasome - alphaL81Y mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: Proteasome subunit alpha

MacromoleculeName: Proteasome subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 25.879465 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQQGQMAYDR AITVFSPDGR LFQVEYAREA VKKGSTALGM KFANGVLLIS DKKVRSRLIE QNSIEKIQLI DDYVAAVTSG YVADARVLV DFARISAQQE KVTYGSLVNI ENLVKRVADQ MQQYTQYGGV RPYGVSLIFA GIDQIGPRLF DCDPAGTINE Y KATAIGSG ...String:
MQQGQMAYDR AITVFSPDGR LFQVEYAREA VKKGSTALGM KFANGVLLIS DKKVRSRLIE QNSIEKIQLI DDYVAAVTSG YVADARVLV DFARISAQQE KVTYGSLVNI ENLVKRVADQ MQQYTQYGGV RPYGVSLIFA GIDQIGPRLF DCDPAGTINE Y KATAIGSG KDAVVSFLER EYKENLPEKE AVTLGIKALK SSLEEGEELK APEIASITVG NKYRIYDQEE VKKFL

UniProtKB: Proteasome subunit alpha

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Macromolecule #2: Proteasome subunit beta

MacromoleculeName: Proteasome subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 23.169811 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNQTLETGTT TVGITLKDAV IMATERRVTM ENFIMHKNGK KLFQIDTYTG MTIAGLVGDA QVLVRYMKAE LELYRLQRRV NMPIEAVAT LLSNMLNQVK YMPYMVQLLV GGIDTAPHVF SIDAAGGSVE DIYASTGSGS PFVYGVLESQ YSEKMTVDEG V DLVIRAIS ...String:
MNQTLETGTT TVGITLKDAV IMATERRVTM ENFIMHKNGK KLFQIDTYTG MTIAGLVGDA QVLVRYMKAE LELYRLQRRV NMPIEAVAT LLSNMLNQVK YMPYMVQLLV GGIDTAPHVF SIDAAGGSVE DIYASTGSGS PFVYGVLESQ YSEKMTVDEG V DLVIRAIS AAKQRDSASG GMIDVAVITR KDGYVQLPTD QIESRIRKLG LIL

UniProtKB: Proteasome subunit beta

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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: PDB ENTRY
PDB model - PDB ID:

1ya7

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 131453
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8f66:
Thermoplasma acidophilum 20S proteasome - L81Y mutation in alpha subunit

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