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- PDB-8i6j: The focused refinement of CCT3-PhLP2A from TRiC-PhLP2A complex in... -

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Basic information

Entry
Database: PDB / ID: 8i6j
TitleThe focused refinement of CCT3-PhLP2A from TRiC-PhLP2A complex in the open state
Components
  • Phosducin-like protein 3
  • T-complex protein 1 subunit gamma
KeywordsCHAPERONE / chaperonin complex / cochaperone
Function / homology
Function and homology information


negative regulation of chaperone-mediated protein folding / perinucleolar compartment / zona pellucida receptor complex / 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 / binding of sperm to zona pellucida / positive regulation of telomerase RNA localization to Cajal body / Folding of actin by CCT/TriC ...negative regulation of chaperone-mediated protein folding / perinucleolar compartment / zona pellucida receptor complex / 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 / binding of sperm to zona pellucida / positive regulation of telomerase RNA localization to Cajal body / Folding of actin by CCT/TriC / 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 / negative regulation of ubiquitin-dependent protein catabolic process / regulation of peptidyl-tyrosine phosphorylation / chaperone-mediated protein folding / protein folding chaperone / positive regulation of endothelial cell proliferation / positive regulation of telomere maintenance via telomerase / ATP-dependent protein folding chaperone / positive regulation of angiogenesis / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / unfolded protein binding / protein folding / cell body / actin cytoskeleton organization / angiogenesis / microtubule / cytoskeleton / protein stabilization / positive regulation of gene expression / apoptotic process / 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, gamma 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) ...: / Phosducin, thioredoxin-like domain / Phosducin / T-complex protein 1, gamma 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
ADENOSINE-5'-DIPHOSPHATE / T-complex protein 1 subunit gamma / Phosducin-like protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.82 Å
AuthorsRoh, S.H. / Park, J. / Kim, H. / Lim, S.
Funding support Korea, Republic Of, 1items
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
DepositionJan 28, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Q: Phosducin-like protein 3
C: T-complex protein 1 subunit gamma
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,6913
Polymers88,2642
Non-polymers4271
Water00
1


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

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Components

#1: Protein Phosducin-like protein 3 / PhPL3


Mass: 27650.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PDCL3, PhLP2A / Production host: Escherichia coli (E. coli) / References: UniProt: Q9H2J4
#2: Protein T-complex protein 1 subunit gamma / TCP-1-gamma / CCT-gamma / hTRiC5


Mass: 60613.855 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCT3, CCTG, TRIC5 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49368
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex of TRiC/CCT and PhLP2ACOMPLEX#1-#20RECOMBINANT
2PhLP2A (PDCL3)COMPLEX#11RECOMBINANT
3TRiC/CCTCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
111 MDaNO
22
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
11Escherichia coli (E. coli)562
22Trichoplusia ni (cabbage looper)7111high fivepFastbac-dual
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
21 mMDTTC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 15075
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2.0particle selectioncryoSPARC was used for 2D classification
4cryoSPARC3.2.0CTF correctioncryoSPARC was used for the CTF correction
7UCSF Chimera1.15model fitting
9PHENIX1.19model refinementPhenix was used for refinement
10Coot0.9.6model refinement
11cryoSPARC3.2.0initial Euler assignment
12cryoSPARC3.2.0final Euler assignment
13cryoSPARC3.2.0classification
14cryoSPARC3.2.03D reconstruction
CTF correctionDetails: CTF correction was performed for every micrographs / Type: NONE
Particle selectionNum. of particles selected: 2691733
Details: The initial particle selection after 2D class classification
3D reconstructionResolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 359533 / Details: Focused refinement map for CCT3 and PhLP2A complex / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6NR8

6nr8


Accession code: 6NR8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035360
ELECTRON MICROSCOPYf_angle_d0.6847237
ELECTRON MICROSCOPYf_dihedral_angle_d7.293736
ELECTRON MICROSCOPYf_chiral_restr0.046846
ELECTRON MICROSCOPYf_plane_restr0.004921

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