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Entry
Database: PDB / ID: 9d0t
TitleCore particle assembly intermediate 1 purified from Saccharomyces cerevisiae
Components
  • (Proteasome subunit alpha type- ...) x 6
  • (Proteasome subunit beta type- ...) x 3
  • Probable proteasome subunit alpha type-7
  • Proteasome activator BLM10
  • Proteasome maturation factor UMP1,Myosin light chain kinase 2, skeletal/cardiac muscle
KeywordsHYDROLASE / Proteasome / assembly chaperone / Blm10 / PA200 / Blm10-13S / Pba1 / CP
Function / homology
Function and homology information


proteasome core complex import into nucleus / myosin-light-chain kinase / myosin light chain kinase activity / myosin light chain binding / proteasome storage granule assembly / peptidase activator activity / cardiac muscle tissue morphogenesis / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Proteasome assembly ...proteasome core complex import into nucleus / myosin-light-chain kinase / myosin light chain kinase activity / myosin light chain binding / proteasome storage granule assembly / peptidase activator activity / cardiac muscle tissue morphogenesis / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Proteasome assembly / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / proteasomal ubiquitin-independent protein catabolic process / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / Regulation of PTEN stability and activity / CDK-mediated phosphorylation and removal of Cdc6 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / KEAP1-NFE2L2 pathway / Neddylation / proteasome binding / Orc1 removal from chromatin / MAPK6/MAPK4 signaling / proteasome storage granule / Antigen processing: Ubiquitination & Proteasome degradation / proteasome endopeptidase complex / Ub-specific processing proteases / endopeptidase activator activity / proteasome core complex, beta-subunit complex / proteasome assembly / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / striated muscle contraction / regulation of proteasomal protein catabolic process / Neutrophil degranulation / : / proteasome complex / ubiquitin-dependent protein catabolic process / endopeptidase activity / proteasome-mediated ubiquitin-dependent protein catabolic process / calmodulin binding / DNA repair / mRNA binding / DNA damage response / endoplasmic reticulum membrane / signal transduction / mitochondrion / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Proteasome activator Blm10, N-terminal / Proteasome-substrate-size regulator, N-terminal / Myosin light chain kinase 2, catalytic domain / Proteasome activator complex subunit 4 C-terminal domain / Proteasome activator Blm10, mid region / Proteasome activator complex subunit 4 / : / Proteasome activator complex subunit 4-like, C-terminal / Proteasome activator complex subunit 4, mid HEAT repeats region / Proteasome activator complex subunit 4-like, HEAT repeat-like ...Proteasome activator Blm10, N-terminal / Proteasome-substrate-size regulator, N-terminal / Myosin light chain kinase 2, catalytic domain / Proteasome activator complex subunit 4 C-terminal domain / Proteasome activator Blm10, mid region / Proteasome activator complex subunit 4 / : / Proteasome activator complex subunit 4-like, C-terminal / Proteasome activator complex subunit 4, mid HEAT repeats region / Proteasome activator complex subunit 4-like, HEAT repeat-like / Proteasome maturation factor Ump1 / Proteasome maturation factor UMP1 / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / 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 / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Myosin light chain kinase 2, skeletal/cardiac muscle / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-2 / Proteasome subunit beta type-3 / Proteasome subunit alpha type-5 / Proteasome maturation factor UMP1 ...Myosin light chain kinase 2, skeletal/cardiac muscle / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-2 / Proteasome subunit beta type-3 / Proteasome subunit alpha type-5 / Proteasome maturation factor UMP1 / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / Proteasome activator BLM10
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsChen, X. / Kaur, M. / Roelofs, J. / Walters, K.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)1 ZIA BC011490 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM149314 United States
CitationJournal: bioRxiv / Year: 2024
Title: Structure of Blm10:13S proteasome intermediate reveals parallel assembly pathways for the proteasome core particle.
Authors: Mandeep Kaur / Xiang Chen / Stella Y Lee / Tyler M Weaver / Bret D Freudenthal / Kylie J Walters / Jeroen Roelofs /
Abstract: Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be ...Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be replaced by Blm10 to form Blm10:CP, which promotes ATP-independent degradation of disordered proteins. Here, we present evidence of distinct parallel assembly pathways for CP by solving five cryo-EM structures including a Blm10:13S pre-assembly intermediate. Our data conflict with the current model of Blm10 and Pba1/Pba2 sequential activity in a single assembly pathway, as we find their CP binding is mutually exclusive and both are present on early and late assembly intermediates. CP affinity for Pba1/Pba2 is reduced during maturation, promoting Pba1/Pba2 release. We find Blm10 undergoes no such affinity switch, suggesting this pathway predominantly yields mature Blm10-bound CP. Altogether, our findings conflict with the current paradigm of sequential CP binding to instead indicate parallel assembly pathways by Pba1/Pba2 and Blm10.
History
DepositionAug 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Proteasome subunit alpha type-1
B: Proteasome subunit alpha type-2
C: Proteasome subunit alpha type-3
D: Proteasome subunit alpha type-4
E: Proteasome subunit alpha type-5
F: Proteasome subunit alpha type-6
G: Probable proteasome subunit alpha type-7
I: Proteasome subunit beta type-2
J: Proteasome subunit beta type-3
K: Proteasome subunit beta type-4
O: Proteasome activator BLM10
P: Proteasome maturation factor UMP1,Myosin light chain kinase 2, skeletal/cardiac muscle


Theoretical massNumber of molelcules
Total (without water)540,82012
Polymers540,82012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Proteasome subunit alpha type- ... , 6 types, 6 molecules ABCDEF

#1: Protein Proteasome subunit alpha type-1 / Macropain subunit C7-alpha / Multicatalytic endopeptidase complex C7 / Proteasome component C7- ...Macropain subunit C7-alpha / Multicatalytic endopeptidase complex C7 / Proteasome component C7-alpha / Proteasome component Y8 / Proteinase YSCE subunit 7 / SCL1 suppressor protein


Mass: 28033.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SCL1, PRC2, PRS2, YGL011C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P21243
#2: Protein Proteasome subunit alpha type-2 / Macropain subunit Y7 / Multicatalytic endopeptidase complex subunit Y7 / Proteasome component Y7 / ...Macropain subunit Y7 / Multicatalytic endopeptidase complex subunit Y7 / Proteasome component Y7 / Proteinase YSCE subunit 7


Mass: 27191.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRE8, PRS4, YML092C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P23639
#3: Protein Proteasome subunit alpha type-3 / Macropain subunit Y13 / Multicatalytic endopeptidase complex subunit Y13 / Proteasome component Y13 ...Macropain subunit Y13 / Multicatalytic endopeptidase complex subunit Y13 / Proteasome component Y13 / Proteinase YSCE subunit 13


Mass: 28748.230 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRE9, PRS5, YGR135W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P23638
#4: Protein Proteasome subunit alpha type-4 / Macropain subunit PRE6 / Multicatalytic endopeptidase complex subunit PRE6 / Proteasome component ...Macropain subunit PRE6 / Multicatalytic endopeptidase complex subunit PRE6 / Proteasome component PRE6 / Proteinase YSCE subunit PRE6


Mass: 28478.111 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRE6, YOL038W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40303
#5: Protein Proteasome subunit alpha type-5 / Macropain subunit PUP2 / Multicatalytic endopeptidase complex subunit PUP2 / Proteasome component ...Macropain subunit PUP2 / Multicatalytic endopeptidase complex subunit PUP2 / Proteasome component PUP2 / Proteinase YSCE subunit PUP2


Mass: 28649.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PUP2, DOA5, YGR253C, G9155 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32379
#6: Protein Proteasome subunit alpha type-6 / Macropain subunit PRE5 / Multicatalytic endopeptidase complex subunit PRE5 / Proteasome component ...Macropain subunit PRE5 / Multicatalytic endopeptidase complex subunit PRE5 / Proteasome component PRE5 / Proteinase YSCE subunit PRE5


Mass: 25634.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRE5, YMR314W, YM9924.06 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40302

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Protein , 3 types, 3 molecules GOP

#7: Protein Probable proteasome subunit alpha type-7 / Macropain subunit C1 / Multicatalytic endopeptidase complex subunit C1 / Proteasome component C1 / ...Macropain subunit C1 / Multicatalytic endopeptidase complex subunit C1 / Proteasome component C1 / Proteinase YSCE subunit 1


Mass: 31575.068 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRE10, PRC1, PRS1, YOR362C, O6650 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P21242
#11: Protein Proteasome activator BLM10 / Bleomycin resistance protein BLM10


Mass: 246282.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: BLM10, BLM3, YFL007W, YFL006W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P43583
#12: Protein Proteasome maturation factor UMP1,Myosin light chain kinase 2, skeletal/cardiac muscle / MLCK2


Mass: 22753.646 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: UMP1, YBR173C, YBR1234, MYLK2 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P38293, UniProt: A4IFM7, myosin-light-chain kinase

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Proteasome subunit beta type- ... , 3 types, 3 molecules IJK

#8: Protein Proteasome subunit beta type-2 / Macropain subunit PUP1 / Multicatalytic endopeptidase complex subunit PUP1 / Proteasome component ...Macropain subunit PUP1 / Multicatalytic endopeptidase complex subunit PUP1 / Proteasome component PUP1 / Proteinase YSCE subunit PUP1


Mass: 28299.889 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PUP1, YOR157C / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P25043, proteasome endopeptidase complex
#9: Protein Proteasome subunit beta type-3 / Macropain subunit PUP3 / Multicatalytic endopeptidase complex subunit PUP3 / Proteasome component PUP3


Mass: 22627.842 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PUP3, YER094C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25451
#10: Protein Proteasome subunit beta type-4 / Macropain subunit C11 / Multicatalytic endopeptidase complex subunit C11 / Proteasome component C11 ...Macropain subunit C11 / Multicatalytic endopeptidase complex subunit C11 / Proteasome component C11 / Proteinase YSCE subunit 11


Mass: 22545.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PRE1, YER012W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P22141

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Details

Has protein modificationN

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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 assembly intermediate / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.534 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Details: 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EGTA, 1 mM DDT
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris(hydroxymethyl)aminomethane hydrochlorideC4H12NO3Cl1
25 mMMagnesium dichlorideMgCl21
32 mMEGTAC14H24N2O101
41 mMDichlorodiphenyltrichloroethaneC14H9Cl51
SpecimenConc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 0.42 mBar, 15mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 298.15 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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.034 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 58
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
2EPUimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraX1.8model fitting
8Coot0.9.8.93 ELmodel fitting
10cryoSPARC4.4.1initial Euler assignment
11cryoSPARC4.4.1final Euler assignment
12cryoSPARC4.4.1classification
13cryoSPARC4.4.13D reconstruction
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4058518
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 586158 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 120.13 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
14V7O

4v7o

CA4V7OCA1
24V7O

4v7o

DA4V7ODA1
34V7O

4v7o

EA4V7OEA1
47LSX

7lsx

A7LSXA2
57LSX

7lsx

B7LSXB2
67LSX

7lsx

C7LSXC2
77LSX

7lsx

D7LSXD2
87LSX

7lsx

E7LSXE2
97LSX

7lsx

F7LSXF2
107LSX

7lsx

G7LSXG2
117LSX

7lsx

H7LSXH2
127LSX

7lsx

I7LSXI2
137LSX

7lsx

J7LSXJ2
147LSX

7lsx

K7LSXK2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01434210
ELECTRON MICROSCOPYf_angle_d1.88346293
ELECTRON MICROSCOPYf_dihedral_angle_d13.68112666
ELECTRON MICROSCOPYf_chiral_restr0.1035259
ELECTRON MICROSCOPYf_plane_restr0.0125919

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