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- EMDB-35280: The focused refinement of CCT4-PhLP2A from TRiC-PhLP2A complex in... -

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Basic information

Entry
Database: EMDB / ID: EMD-35280
TitleThe focused refinement of CCT4-PhLP2A from TRiC-PhLP2A complex in the open state
Map dataA focused refined map of CCT4-PhLP2A
Sample
  • Complex: Complex of human TRiC/CCT-PhLP2A
    • Complex: T-complex protein 1 subunit delta from human TRiC/CCT complex
      • Protein or peptide: T-complex protein 1 subunit delta
    • Complex: PhLP2A (PDCL3)
      • Protein or peptide: Phosducin-like protein 3
Keywordschaperonin complex / CHAPERONE / cochaperone
Function / homology
Function and homology information


negative regulation of chaperone-mediated protein folding / perinucleolar compartment / zona pellucida receptor complex / scaRNA localization to Cajal body / positive regulation of establishment of protein localization to telomere / positive regulation of protein localization to Cajal body / BBSome-mediated cargo-targeting to cilium / chaperonin-containing T-complex / Folding of actin by CCT/TriC / binding of sperm to zona pellucida ...negative regulation of chaperone-mediated protein folding / perinucleolar compartment / zona pellucida receptor complex / scaRNA localization to Cajal body / positive regulation of establishment of protein localization to telomere / positive regulation of protein localization to Cajal body / BBSome-mediated cargo-targeting to cilium / chaperonin-containing T-complex / Folding of actin by CCT/TriC / binding of sperm to zona pellucida / positive regulation of telomerase RNA localization to Cajal body / Formation of tubulin folding intermediates by CCT/TriC / vascular endothelial growth factor receptor 2 binding / Prefoldin mediated transfer of substrate to CCT/TriC / Association of TriC/CCT with target proteins during biosynthesis / chaperone-mediated protein folding / protein folding chaperone / regulation of peptidyl-tyrosine phosphorylation / positive regulation of telomerase activity / positive regulation of endothelial cell proliferation / positive regulation of telomere maintenance via telomerase / negative regulation of ubiquitin-dependent protein catabolic process / cell projection / ATP-dependent protein folding chaperone / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / positive regulation of angiogenesis / unfolded protein binding / melanosome / protein folding / cell body / actin cytoskeleton organization / angiogenesis / microtubule / protein stabilization / centrosome / apoptotic process / positive regulation of gene expression / perinuclear region of cytoplasm / endoplasmic reticulum / ATP hydrolysis activity / protein-containing complex / RNA binding / extracellular exosome / nucleoplasm / ATP binding / cytosol / cytoplasm
Similarity search - Function
Phosducin, thioredoxin-like domain / Phosducin / T-complex protein 1, delta subunit / Chaperonins TCP-1 signature 1. / Chaperonins TCP-1 signature 2. / Chaperonin TCP-1, conserved site / Chaperonins TCP-1 signature 3. / Chaperone tailless complex polypeptide 1 (TCP-1) / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily ...Phosducin, thioredoxin-like domain / Phosducin / T-complex protein 1, delta subunit / Chaperonins TCP-1 signature 1. / Chaperonins TCP-1 signature 2. / Chaperonin TCP-1, conserved site / Chaperonins TCP-1 signature 3. / Chaperone tailless complex polypeptide 1 (TCP-1) / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / Thioredoxin-like superfamily
Similarity search - Domain/homology
T-complex protein 1 subunit delta / Phosducin-like protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.22 Å
AuthorsRoh SH / Park J / Kim H / Lim S
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea) Korea, Republic Of
CitationJournal: Nat Commun / Year: 2024
Title: A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.
Authors: Junsun Park / Hyunmin Kim / Daniel Gestaut / Seyeon Lim / Kwadwo A Opoku-Nsiah / Alexander Leitner / Judith Frydman / Soung-Hun Roh /
Abstract: Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin- ...Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.
History
DepositionFeb 7, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35280.png

Downloads & links

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Map

FileDownload / File: emd_35280.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA focused refined map of CCT4-PhLP2A
Voxel sizeX=Y=Z: 1.13 Å
Density
Contour LevelBy AUTHOR: 0.616
Minimum - Maximum-4.2455406 - 6.842855
Average (Standard dev.)0.0032142631 (±0.059223834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 361.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: A focused refined half map of CCT4-PhLP2A

Fileemd_35280_half_map_1.map
AnnotationA focused refined half map of CCT4-PhLP2A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: A focused refined half map of CCT4-PhLP2A

Fileemd_35280_half_map_2.map
AnnotationA focused refined half map of CCT4-PhLP2A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of human TRiC/CCT-PhLP2A

EntireName: Complex of human TRiC/CCT-PhLP2A
Components
  • Complex: Complex of human TRiC/CCT-PhLP2A
    • Complex: T-complex protein 1 subunit delta from human TRiC/CCT complex
      • Protein or peptide: T-complex protein 1 subunit delta
    • Complex: PhLP2A (PDCL3)
      • Protein or peptide: Phosducin-like protein 3

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Supramolecule #1: Complex of human TRiC/CCT-PhLP2A

SupramoleculeName: Complex of human TRiC/CCT-PhLP2A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 1 MDa

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Supramolecule #2: T-complex protein 1 subunit delta from human TRiC/CCT complex

SupramoleculeName: T-complex protein 1 subunit delta from human TRiC/CCT complex
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: PhLP2A (PDCL3)

SupramoleculeName: PhLP2A (PDCL3) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: T-complex protein 1 subunit delta

MacromoleculeName: T-complex protein 1 subunit delta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.996113 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MPENVAPRSG ATAGAAGGRG KGAYQDRDKP AQIRFSNISA AKAVADAIRT SLGPKGMDKM IQDGKGDVTI TNDGATILKQ MQVLHPAAR MLVELSKAQD IEAGDGTTSV VIIAGSLLDS CTKLLQKGIH PTIISESFQK ALEKGIEILT DMSRPVELSD R ETLLNSAT ...String:
MPENVAPRSG ATAGAAGGRG KGAYQDRDKP AQIRFSNISA AKAVADAIRT SLGPKGMDKM IQDGKGDVTI TNDGATILKQ MQVLHPAAR MLVELSKAQD IEAGDGTTSV VIIAGSLLDS CTKLLQKGIH PTIISESFQK ALEKGIEILT DMSRPVELSD R ETLLNSAT TSLNSKVVSQ YSSLLSPMSV NAVMKVIDPA TATSVDLRDI KIVKKLGGTI DDCELVEGLV LTQKVSNSGI TR VEKAKIG LIQFCLSAPK TDMDNQIVVS DYAQMDRVLR EERAYILNLV KQIKKTGCNV LLIQKSILRD ALSDLALHFL NKM KIMVIK DIEREDIEFI CKTIGTKPVA HIDQFTADML GSAELAEEVN LNGSGKLLKI TGCASPGKTV TIVVRGSNKL VIEE AERSI HDALCVIRCL VKKRALIAGG GAPEIELALR LTEYSRTLSG MESYCVRAFA DAMEVIPSTL AENAGLNPIS TVTEL RNRH AQGEKTAGIN VRKGGISNIL EELVVQPLLV SVSALTLATE TVRSILKIDD VVNTR

UniProtKB: T-complex protein 1 subunit delta

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Macromolecule #2: Phosducin-like protein 3

MacromoleculeName: Phosducin-like protein 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.650383 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQDPNADTEW NDILRKKGIL PPKESLKELE EEAEEEQRIL QQSVVKTYED MTLEELEDHE DEFNEEDERA IEMYRRRRLA EWKATKLKN KFGEVLEISG KDYVQEVTKA GEGLWVILHL YKQGIPLCAL INQHLSGLAR KFPDVKFIKA ISTTCIPNYP D RNLPTIFV ...String:
MQDPNADTEW NDILRKKGIL PPKESLKELE EEAEEEQRIL QQSVVKTYED MTLEELEDHE DEFNEEDERA IEMYRRRRLA EWKATKLKN KFGEVLEISG KDYVQEVTKA GEGLWVILHL YKQGIPLCAL INQHLSGLAR KFPDVKFIKA ISTTCIPNYP D RNLPTIFV YLEGDIKAQF IGPLVFGGMN LTRDELEWKL SESGAIMTDL EENPKKPIED VLLSSVRRSV LMKRDSDSEG D

UniProtKB: Phosducin-like protein 3

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chloridesodium chloride
1.0 mMC4H10O2S2DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 15075 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2691733
Details: The initial particle selection after 2D class classification
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6nra


Details: The model of TRiC/CCT complex from the previous study
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationNumber classes: 5 / Avg.num./class: 500000 / Software - Name: cryoSPARC (ver. 3.2.0) / Details: The final 3D classification
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 485964
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6nr8


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8i9q:
The focused refinement of CCT4-PhLP2A from TRiC-PhLP2A complex in the open state

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