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- EMDB-35199: The focused refinement of CCT3-PhLP2A from TRiC-PhLP2A complex in... -

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Basic information

Entry
Database: EMDB / ID: EMD-35199
TitleThe focused refinement of CCT3-PhLP2A from TRiC-PhLP2A complex in the open state
Map data
Sample
  • Complex: Complex of TRiC/CCT and PhLP2A
    • Complex: PhLP2A (PDCL3)
      • Protein or peptide: Phosducin-like protein 3
    • Complex: TRiC/CCT
      • Protein or peptide: T-complex protein 1 subunit gamma
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Keywordschaperonin complex / CHAPERONE / cochaperone
Function / homology
Function and homology information


: / perinucleolar compartment / zona pellucida receptor complex / positive regulation of protein localization to Cajal body / positive regulation of telomerase RNA localization to Cajal body / chaperonin-containing T-complex / : / BBSome-mediated cargo-targeting to cilium / Formation of tubulin folding intermediates by CCT/TriC / binding of sperm to zona pellucida ...: / perinucleolar compartment / zona pellucida receptor complex / positive regulation of protein localization to Cajal body / positive regulation of telomerase RNA localization to Cajal body / chaperonin-containing T-complex / : / BBSome-mediated cargo-targeting to cilium / Formation of tubulin folding intermediates by CCT/TriC / binding of sperm to zona pellucida / Folding of actin by CCT/TriC / vascular endothelial growth factor receptor 2 binding / Prefoldin mediated transfer of substrate to CCT/TriC / regulation of peptidyl-tyrosine phosphorylation / Association of TriC/CCT with target proteins during biosynthesis / negative regulation of ubiquitin-dependent protein catabolic process / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / : / positive regulation of telomere maintenance via telomerase / positive regulation of endothelial cell proliferation / protein folding chaperone / 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 / apoptotic process / positive regulation of gene expression / perinuclear region of cytoplasm / endoplasmic reticulum / protein-containing complex / ATP hydrolysis activity / RNA binding / extracellular exosome / nucleoplasm / ATP binding / cytoplasm / cytosol
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. ...: / 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
T-complex protein 1 subunit gamma / Phosducin-like protein 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.82 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
DepositionJan 28, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35199.png

Downloads & links

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Map

FileDownload / File: emd_35199.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.13 Å/pix.
x 320 pix.
= 361.6 Å		1.13 Å/pix.
x 320 pix.
= 361.6 Å		1.13 Å/pix.
x 320 pix.
= 361.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.13 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.639
Minimum - Maximum-3.795292 - 6.498461
Average (Standard dev.)0.0033117947 (±0.07161466)
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 half map for PhLP2A-CCT3 open TRiC focused map

Fileemd_35199_half_map_1.map
AnnotationA half map for PhLP2A-CCT3 open TRiC focused map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: A half map for PhLP2A-CCT3 open TRiC focused map

Fileemd_35199_half_map_2.map
AnnotationA half map for PhLP2A-CCT3 open TRiC focused map
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 TRiC/CCT and PhLP2A

EntireName: Complex of TRiC/CCT and PhLP2A
Components
  • Complex: Complex of TRiC/CCT and PhLP2A
    • Complex: PhLP2A (PDCL3)
      • Protein or peptide: Phosducin-like protein 3
    • Complex: TRiC/CCT
      • Protein or peptide: T-complex protein 1 subunit gamma
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Complex of TRiC/CCT and PhLP2A

SupramoleculeName: Complex of TRiC/CCT and PhLP2A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1 MDa

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Supramolecule #2: PhLP2A (PDCL3)

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

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Supramolecule #3: TRiC/CCT

SupramoleculeName: TRiC/CCT / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Phosducin-like protein 3

MacromoleculeName: Phosducin-like protein 3 / type: protein_or_peptide / ID: 1 / 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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Macromolecule #2: T-complex protein 1 subunit gamma

MacromoleculeName: T-complex protein 1 subunit gamma / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.613855 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MMGHRPVLVL SQNTKRESGR KVQSGNINAA KTIADIIRTC LGPKSMMKML LDPMGGIVMT NDGNAILREI QVQHPAAKSM IEISRTQDE EVGDGTTSVI ILAGEMLSVA EHFLEQQMHP TVVISAYRKA LDDMISTLKK ISIPVDISDS DMMLNIINSS I TTKAISRW ...String:
MMGHRPVLVL SQNTKRESGR KVQSGNINAA KTIADIIRTC LGPKSMMKML LDPMGGIVMT NDGNAILREI QVQHPAAKSM IEISRTQDE EVGDGTTSVI ILAGEMLSVA EHFLEQQMHP TVVISAYRKA LDDMISTLKK ISIPVDISDS DMMLNIINSS I TTKAISRW SSLACNIALD AVKMVQFEEN GRKEIDIKKY ARVEKIPGGI IEDSCVLRGV MINKDVTHPR MRRYIKNPRI VL LDSSLEY KKGESQTDIE ITREEDFTRI LQMEEEYIQQ LCEDIIQLKP DVVITEKGIS DLAQHYLMRA NITAIRRVRK TDN NRIARA CGARIVSRPE ELREDDVGTG AGLLEIKKIG DEYFTFITDC KDPKACTILL RGASKEILSE VERNLQDAMQ VCRN VLLDP QLVPGGGASE MAVAHALTEK SKAMTGVEQW PYRAVAQALE VIPRTLIQNC GASTIRLLTS LRAKHTQENC ETWGV NGET GTLVDMKELG IWEPLAVKLQ TYKTAVETAV LLLRIDDIVS GHKKKGDDQS RQGGAPDAGQ E

UniProtKB: T-complex protein 1 subunit gamma

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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 chloride
1.0 mMC4H10O2S2DTT
GridModel: Quantifoil R1.2/1.3 / 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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
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
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Details: Focused refinement map for CCT3 and PhLP2A complex / Number images used: 359533
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final 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
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-8i6j:
The focused refinement of CCT3-PhLP2A from TRiC-PhLP2A complex in the open state

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