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- PDB-6muv: The structure of the Plasmodium falciparum 20S proteasome in comp... -

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Basic information

Entry
Database: PDB / ID: 6muv
TitleThe structure of the Plasmodium falciparum 20S proteasome in complex with two PA28 activators
Components
  • (20S proteasome alpha- ...) x 7
  • (20S proteasome beta- ...) x 7
  • Proteasome activator PA28
KeywordsHYDROLASE / proteasome / protease / 11S subunit / hydrolyse activator / proteasome activator / complex
Function / homology
Function and homology information


Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / UCH proteinases / positive regulation of endopeptidase activity / proteasome activator complex / regulation of G1/S transition of mitotic cell cycle / endopeptidase activator activity / proteasome core complex, beta-subunit complex / proteasome core complex ...Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / UCH proteinases / positive regulation of endopeptidase activity / proteasome activator complex / regulation of G1/S transition of mitotic cell cycle / endopeptidase activator activity / proteasome core complex, beta-subunit complex / proteasome core complex / proteasomal ubiquitin-independent protein catabolic process / proteasome endopeptidase complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / regulation of proteasomal protein catabolic process / proteasomal protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / peptidase activity / ubiquitin-dependent protein catabolic process / endopeptidase activity / nucleoplasm / nucleus / cytoplasm
Proteasome subunit alpha 1 / Proteasome subunit alpha5 / Proteasome activator PA28 / Proteasome beta-type subunit, conserved site / Proteasome subunit beta 4 / Proteasome alpha-type subunit / Proteasome B-type subunit / Nucleophile aminohydrolases, N-terminal / Proteasome beta 3 subunit / Proteasome subunit alpha6 ...Proteasome subunit alpha 1 / Proteasome subunit alpha5 / Proteasome activator PA28 / Proteasome beta-type subunit, conserved site / Proteasome subunit beta 4 / Proteasome alpha-type subunit / Proteasome B-type subunit / Nucleophile aminohydrolases, N-terminal / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome, subunit alpha/beta / Proteasome subunit alpha2 / Proteasome subunit / Proteasome subunit beta 1 / Proteasome subunit beta 2 / Proteasome subunit beta 7 / Proteasome activator superfamily / Proteasome activator PA28, C-terminal domain superfamily / Proteasome activator PA28, C-terminal domain / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit beta Pre3 / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit profile. / Proteasome alpha-type subunit profile. / Proteasome beta-type subunits signature. / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome activator pa28 beta subunit / Proteasome subunit alpha 3
Proteasome endopeptidase complex / Proteasome endopeptidase complex / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Subunit of proteaseome activator complex,putative / Proteasome subunit beta ...Proteasome endopeptidase complex / Proteasome endopeptidase complex / Proteasome subunit beta / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Subunit of proteaseome activator complex,putative / Proteasome subunit beta / Proteasome subunit beta type-6, putative / Proteasome subunit beta / Proteasome endopeptidase complex / Proteasome subunit beta / Proteasome subunit beta
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsMetcalfe, R.D. / Xie, S.C. / Hanssen, E. / Gillett, D.L. / Leis, A.P. / Tilley, L. / Griffin, M.D.W.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (Australia) Australia
Australian Research Council Australia
CitationJournal: Nat Microbiol / Year: 2019
Title: The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.
Authors: Stanley C Xie / Riley D Metcalfe / Eric Hanssen / Tuo Yang / David L Gillett / Andrew P Leis / Craig J Morton / Michael J Kuiper / Michael W Parker / Natalie J Spillman / Wilson Wong / Christopher Tsu / Lawrence R Dick / Michael D W Griffin / Leann Tilley /
Abstract: The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its ...The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Movie
  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: 20S proteasome alpha-1 subunit
B: 20S proteasome alpha-2 subunit
C: 20S proteasome alpha-3 subunit
D: 20S proteasome alpha-4 subunit
E: 20S proteasome alpha-5 subunit
F: 20S proteasome alpha-6 subunit
G: 20S proteasome alpha-7 subunit
H: 20S proteasome beta-1 subunit
I: 20S proteasome beta-2 subunit
J: 20S proteasome beta-3 subunit
K: 20S proteasome beta-4 subunit
L: 20S proteasome beta-5 subunit
M: 20S proteasome beta-6 subunit
N: 20S proteasome beta-7 subunit
P: 20S proteasome alpha-2 subunit
R: 20S proteasome alpha-4 subunit
S: 20S proteasome alpha-5 subunit
V: 20S proteasome beta-1 subunit
W: 20S proteasome beta-2 subunit
Y: 20S proteasome beta-4 subunit
a: 20S proteasome beta-6 subunit
c: Proteasome activator PA28
d: Proteasome activator PA28
e: Proteasome activator PA28
f: Proteasome activator PA28
g: Proteasome activator PA28
h: Proteasome activator PA28
i: Proteasome activator PA28
j: Proteasome activator PA28
k: Proteasome activator PA28
l: Proteasome activator PA28
m: Proteasome activator PA28
n: Proteasome activator PA28
o: Proteasome activator PA28
p: Proteasome activator PA28
b: 20S proteasome beta-7 subunit
X: 20S proteasome beta-3 subunit
Q: 20S proteasome alpha-3 subunit
T: 20S proteasome alpha-6 subunit
O: 20S proteasome alpha-1 subunit
Z: 20S proteasome beta-5 subunit
U: 20S proteasome alpha-7 subunit


Theoretical massNumber of molelcules
Total (without water)1,227,39342
Polymers1,227,39342
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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20S proteasome alpha- ... , 7 types, 14 molecules AOBPCQDRESFTGU

#1: Protein/peptide 20S proteasome alpha-1 subunit


Mass: 29531.656 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IAR3, proteasome endopeptidase complex
#2: Protein/peptide 20S proteasome alpha-2 subunit


Mass: 26556.391 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: C6KST3, proteasome endopeptidase complex
#3: Protein/peptide 20S proteasome alpha-3 subunit


Mass: 27977.664 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IDG3, proteasome endopeptidase complex
#4: Protein/peptide 20S proteasome alpha-4 subunit


Mass: 27263.285 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IDG2, proteasome endopeptidase complex
#5: Protein/peptide 20S proteasome alpha-5 subunit


Mass: 28417.367 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IBI3, proteasome endopeptidase complex
#6: Protein/peptide 20S proteasome alpha-6 subunit


Mass: 28871.697 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IK90, proteasome endopeptidase complex
#7: Protein/peptide 20S proteasome alpha-7 subunit


Mass: 29324.295 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: O77396, proteasome endopeptidase complex

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20S proteasome beta- ... , 7 types, 14 molecules HVIWJXKYLZMaNb

#8: Protein/peptide 20S proteasome beta-1 subunit


Mass: 29143.936 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8I0U7, proteasome endopeptidase complex
#9: Protein/peptide 20S proteasome beta-2 subunit


Mass: 25104.885 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8I6T3, proteasome endopeptidase complex
#10: Protein/peptide 20S proteasome beta-3 subunit


Mass: 24533.131 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8I261, proteasome endopeptidase complex
#11: Protein/peptide 20S proteasome beta-4 subunit


Mass: 22889.105 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IKC9, proteasome endopeptidase complex
#12: Protein/peptide 20S proteasome beta-5 subunit


Mass: 23620.646 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q8IJT1, proteasome endopeptidase complex
#13: Protein/peptide 20S proteasome beta-6 subunit


Mass: 27301.203 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: C0H4E8, proteasome endopeptidase complex
#14: Protein/peptide 20S proteasome beta-7 subunit


Mass: 30909.893 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7
References: UniProt: Q7K6A9, proteasome endopeptidase complex

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Protein/peptide , 1 types, 14 molecules cdefghijklmnop

#15: Protein/peptide
Proteasome activator PA28


Mass: 33178.773 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (isolate 3D7) (eukaryote)
Strain: isolate 3D7 / Gene: PF3D7_0907700 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8I374

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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

Component

Type: COMPLEX

IDNameDetailsEntity IDParent-IDSource
120S proteasome/PA28 complex.Sample was a mixture of unbound 20S proteasome, 20S proteasome in complex with one PA28 cap, and 20S proteasome in complex with two PA28 caps.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 150MULTIPLE SOURCES
2Plasmodium falciparum 20S proteasome1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 141NATURAL
311S activator of the Plasmodium falciparum proteasome.151RECOMBINANT
Molecular weight

Entity assembly-ID: 1 / Experimental value: NO

IDValue (°)
11.22 MDa
20.76 MDa
30.23 MDa
Source (natural)

Ncbi tax-ID: 36329 / Organism: Plasmodium falciparum 3D7 (eukaryote)

IDEntity assembly-ID
21
32
43
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.4
Buffer component

Buffer-ID: 1

IDConc.NameFormula
1100 mMSodium ChlorideNaClSodium chloride
25 mMMagnesium ChlorideMgCl2
31 mMDithiothreitolDTT
425 mMTris hydrochlorideTris-HClTris
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: wait time 0sec blot time 2sec blot force -1

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 100000 X / Nominal defocus min: 1000 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 32 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5200
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µns / Width: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION2.1particle selectionmanual pick then autopick
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION33D reconstruction
19PHENIX1.14model refinement
Image processingDetails: Images were gain corrected
CTF correctionType: NONE
Particle selectionNum. of particles selected: 212749
Details: Relion autopick from 5 class averages resulting from 200o0 particle picked manually
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27516 / Symmetry type: POINT
Atomic model buildingB value: 99.23 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6DFK

6dfk


Pdb chain-ID: M

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