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- EMDB-37344: Local refinement cryo-EM map of human 26S RP (Ed state) bound to ... -

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Entry
Database: EMDB / ID: EMD-37344
TitleLocal refinement cryo-EM map of human 26S RP (Ed state) bound to K11/K48-branched ubiquitin (Ub) chain composed of four Ub, focused on AAA+ ATPase subcomplex.
Map dataLocal refinement cryo-EM map of human 26S RP (Ed state) bound to K11/K48-branched ubiquitin (Ub) chain composed of four Ub, focused on AAA ATPase subcomplex.
Sample
  • Complex: Human 26S proteasome
    • Complex: 19S regulatory particle (RP) of human 26S proteasome
      • Complex: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome
    • Complex: alpha subunits of the 20S core particle (CP) of human 26S proteasome
    • Complex: Ubiquitinated substrate
      • Complex: K11/K48-branched ubiquitin (Ub) chain composed of four Ub.
      • Complex: Sic1_PY substrate polypeptide
Keywordsprotein degradation / macromolecular complex / ubiquitin-proteasome system / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHsu STD / Draczkowski P / Wang YS
Funding support Taiwan, 4 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-CDA-109- L08 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 109-3114-Y-001-001 Taiwan
Ministry of Science and Technology (MoST, Taiwan)MOST 110-2113-M-001- 050-MY3 Taiwan
Ministry of Science and Technology (MoST, Taiwan)110-2311-B-001-013-MY3 Taiwan
CitationJournal: To Be Published
Title: Cryo-EM structure of human 26S proteasomal RP subcomplex bound with branched Ub chain.
Authors: Draczkowski P / Hsu STD / Wang YS
History
DepositionAug 30, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37344.png

Downloads & links

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Map

FileDownload / File: emd_37344.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement cryo-EM map of human 26S RP (Ed state) bound to K11/K48-branched ubiquitin (Ub) chain composed of four Ub, focused on AAA ATPase subcomplex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 400 pix.
= 560. Å		1.4 Å/pix.
x 400 pix.
= 560. Å		1.4 Å/pix.
x 400 pix.
= 560. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.60969204 - 1.9505413
Average (Standard dev.)-0.002522911 (±0.049249317)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 560.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Local refinement cryo-EM half map of human 26S...

Fileemd_37344_half_map_1.map
AnnotationLocal refinement cryo-EM half map of human 26S RP (Ed state) bound to K11/K48-branched ubiquitin (Ub) chain composed of four Ub, focused on AAA ATPase subcomplex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local refinement cryo-EM half map of human 26S...

Fileemd_37344_half_map_2.map
AnnotationLocal refinement cryo-EM half map of human 26S RP (Ed state) bound to K11/K48-branched ubiquitin (Ub) chain composed of four Ub, focused on AAA ATPase subcomplex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human 26S proteasome

EntireName: Human 26S proteasome
Components
  • Complex: Human 26S proteasome
    • Complex: 19S regulatory particle (RP) of human 26S proteasome
      • Complex: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome
    • Complex: alpha subunits of the 20S core particle (CP) of human 26S proteasome
    • Complex: Ubiquitinated substrate
      • Complex: K11/K48-branched ubiquitin (Ub) chain composed of four Ub.
      • Complex: Sic1_PY substrate polypeptide

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Supramolecule #1: Human 26S proteasome

SupramoleculeName: Human 26S proteasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#27
Details: Images of double-capped (RP-CP-RP) 26S proteasome particles were split in half. After combining with subset of single-capped (RP-CP) complexes the signal in particle images was partially ...Details: Images of double-capped (RP-CP-RP) 26S proteasome particles were split in half. After combining with subset of single-capped (RP-CP) complexes the signal in particle images was partially subtracted leaving only the signal of 19S RP together with alpha subunits of the 20S CP proteasomal subcomplex.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #2: 19S regulatory particle (RP) of human 26S proteasome

SupramoleculeName: 19S regulatory particle (RP) of human 26S proteasome / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5, #13-#19, #21-#26
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome

SupramoleculeName: AAA+-ATPase of 19S regulatory particle (RP) of human 26S proteasome
type: complex / ID: 3 / Parent: 2 / Macromolecule list: #1-#5, #21

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Supramolecule #4: alpha subunits of the 20S core particle (CP) of human 26S proteasome

SupramoleculeName: alpha subunits of the 20S core particle (CP) of human 26S proteasome
type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6-#12
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: Ubiquitinated substrate

SupramoleculeName: Ubiquitinated substrate / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #20, #27
Details: Sic1_PY conjugated with K11/K48-branched ubiquitin chain.

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Supramolecule #6: K11/K48-branched ubiquitin (Ub) chain composed of four Ub.

SupramoleculeName: K11/K48-branched ubiquitin (Ub) chain composed of four Ub.
type: complex / ID: 6 / Parent: 5 / Macromolecule list: #27
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #7: Sic1_PY substrate polypeptide

SupramoleculeName: Sic1_PY substrate polypeptide / type: complex / ID: 7 / Parent: 5 / Macromolecule list: #20
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
40.0 mMNaClsodium chloride
5.0 mMMgCl2magnesium chloride
2.0 mMC2H6OS2-Mercaptoethanol
0.5 mMC12H8N21,10-Phenanthroline
2.0 mMC10H16N5O13P3adenosine triphosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: incubation time= 3 s blotting time= 2.5 s.
DetailsThe complex was additionally supplemented with an excess of preformed and SEC-purified RPN13:UCHL5 complex.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
SoftwareName: EPU
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 3 / Number real images: 15242 / Average exposure time: 1.8 sec. / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 70000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2695911
Startup modelType of model: INSILICO MODEL
In silico model: Generated by cryoSPARC using Ab-initio Reconstruction job type.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Software - details: Local Refinment / Number images used: 84476
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Performed in Relion.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Details: Performed in cryoSPARC using Local Focused Refinment job type.
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1)
Details: 3D classification without angular search with mask focused on the AAA+-ATPase of 19S regulatory particle.

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