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- EMDB-5640: cryo-EM structure of CCT5 complex with 1mM ATP/AlFx (C8 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-5640
Titlecryo-EM structure of CCT5 complex with 1mM ATP/AlFx (C8 symmetry)
Map dataC8-symmetry imposed 3D structure of CCT5 complex with 1mM ATP/AlFx
Sample
  • Sample: CCT5 Complex
  • Protein or peptide: chaperonin containing TCP1, subunit 5 (epsilon)
KeywordsTRiC / CCT / chaperonin / protein folding / cryo-electron microscopy
Function / homology
Function and homology information


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 / Prefoldin mediated transfer of substrate to CCT/TriC / beta-tubulin binding ...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 / Prefoldin mediated transfer of substrate to CCT/TriC / beta-tubulin binding / Association of TriC/CCT with target proteins during biosynthesis / chaperone-mediated protein folding / protein folding chaperone / positive regulation of telomere maintenance via telomerase / mRNA 3'-UTR binding / ATP-dependent protein folding chaperone / response to virus / mRNA 5'-UTR binding / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / G-protein beta-subunit binding / unfolded protein binding / protein folding / cell body / microtubule / protein stabilization / centrosome / nucleolus / ATP hydrolysis activity / extracellular exosome / ATP binding / cytosol / cytoplasm
Similarity search - Function
T-complex protein 1, epsilon subunit / T-complex protein 1, epsilon subunit / Chaperonins TCP-1 signature 1. / : / Chaperonins TCP-1 signature 2. / Chaperonin TCP-1, conserved site / Chaperonin TCP-1, conserved site / Chaperonins TCP-1 signature 3. / Chaperone tailless complex polypeptide 1 (TCP-1) / Chaperone tailless complex polypeptide 1 (TCP-1) ...T-complex protein 1, epsilon subunit / T-complex protein 1, epsilon subunit / Chaperonins TCP-1 signature 1. / : / Chaperonins TCP-1 signature 2. / Chaperonin TCP-1, conserved site / Chaperonin TCP-1, conserved site / Chaperonins TCP-1 signature 3. / Chaperone tailless complex polypeptide 1 (TCP-1) / 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 / Chaperonin Cpn60/GroEL/TCP-1 family
Similarity search - Domain/homology
T-complex protein 1 subunit epsilon
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 22.0 Å
AuthorsSergeeva OA / Chen B / Haase-Pettingell C / Ludtke SJ / Chiu W / King JA
CitationJournal: J Biol Chem / Year: 2013
Title: Human CCT4 and CCT5 chaperonin subunits expressed in Escherichia coli form biologically active homo-oligomers.
Authors: Oksana A Sergeeva / Bo Chen / Cameron Haase-Pettingell / Steven J Ludtke / Wah Chiu / Jonathan A King /
Abstract: Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a ...Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.
History
DepositionApr 14, 2013-
Header (metadata) releaseMay 22, 2013-
Map releaseMay 22, 2013-
UpdateJun 26, 2013-
Current statusJun 26, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.35
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5640.png

Downloads & links

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Map

FileDownload / File: emd_5640.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC8-symmetry imposed 3D structure of CCT5 complex with 1mM ATP/AlFx
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 144 pix.
= 311.04 Å		2.16 Å/pix.
x 144 pix.
= 311.04 Å		2.16 Å/pix.
x 144 pix.
= 311.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.35 / Movie #1: 1.35
Minimum - Maximum-0.05033418 - 2.47017598
Average (Standard dev.)0.09533317 (±0.34677681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 311.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z311.040311.040311.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-0.0502.4700.095

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Supplemental data

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Sample components

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Entire : CCT5 Complex

EntireName: CCT5 Complex
Components
  • Sample: CCT5 Complex
  • Protein or peptide: chaperonin containing TCP1, subunit 5 (epsilon)

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Supramolecule #1000: CCT5 Complex

SupramoleculeName: CCT5 Complex / type: sample / ID: 1000 / Oligomeric state: hexadecamer / Number unique components: 1
Molecular weightExperimental: 1 MDa / Theoretical: 960 KDa / Method: size exclusion column (Superose 6 10/300 GL)

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Macromolecule #1: chaperonin containing TCP1, subunit 5 (epsilon)

MacromoleculeName: chaperonin containing TCP1, subunit 5 (epsilon) / type: protein_or_peptide / ID: 1 / Name.synonym: CCT5 / Number of copies: 16 / Oligomeric state: Hexadecamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 1 MDa / Theoretical: 960 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) RIL / Recombinant plasmid: pET21b
SequenceUniProtKB: T-complex protein 1 subunit epsilon
GO: nucleolus, cytoplasm, centrosome, cytosol, chaperonin-containing T-complex
InterPro: T-complex protein 1, epsilon subunit, Chaperone tailless complex polypeptide 1 (TCP-1), Chaperonin TCP-1, conserved site, Chaperonin Cpn60/GroEL/TCP-1 family

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.35 mg/mL
BufferpH: 7.4
Details: 20 mM HEPES/KOH, pH 7.4, 200 mM NaCl, 1 mM DTT, 10 mM MgCl2, 5% glycerol, 5 mM Al(NO3)3, 30 mM NaF, 1 mM ATP
GridDetails: plasma-cleaned R1.2/1.3 Quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
Method: Front side blotting for 4 seconds twice before plunging

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureMin: 94.98 K / Max: 95 K / Average: 94.99 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 250,000 times magnification
Specialist opticsEnergy filter - Name: Gatan in-column energy filter
DetailsMDS mode imaging was used.
DateMar 26, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 160 / Average electron dose: 20 e/Å2 / Camera length: 120
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 71361 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: This holder operates at -178 C (95K). / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Details6,307 particles (ATP-AlFx state) were boxed out semi-automatically using e2boxer.py. The later steps of 3D reconstruction were performed using EMAN1.
CTF correctionDetails: each frame
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: EMAN / Number images used: 2974
Final two d classificationNumber classes: 67

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