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- EMDB-2962: Cryo-electron microscopy structure of the unoccupied population o... -

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Basic information

Entry
Database: EMDB / ID: EMD-2962
TitleCryo-electron microscopy structure of the unoccupied population of CCT5 complexes after incubation with mHtt
Map dataSingle particle tomography reconstruction of unoccupied CCT5 complex after incubation with mutant huntington.
Sample
  • Sample: Unoccupied CCT5 complex after incubation with mutant huntingtin exon 1.
  • Protein or peptide: chaperonin containing TCP1, subunit 5 (epsilon) complex
Keywordschaperonin / Huntington's disease / protein aggregation
Function / homology
Function and homology information


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 / Folding of actin by CCT/TriC / binding of sperm to zona pellucida / Prefoldin mediated transfer of substrate to CCT/TriC / beta-tubulin binding ...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 / Folding of actin by CCT/TriC / binding of sperm to zona pellucida / Prefoldin mediated transfer of substrate to CCT/TriC / beta-tubulin binding / Association of TriC/CCT with target proteins during biosynthesis / : / positive regulation of telomere maintenance via telomerase / protein folding chaperone / 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 / unfolded protein binding / G-protein beta-subunit binding / protein folding / cell body / microtubule / protein stabilization / centrosome / ATP hydrolysis activity / extracellular exosome / ATP binding / cytosol
Similarity search - Function
T-complex protein 1, epsilon 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 ...T-complex protein 1, epsilon 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 / Chaperonin Cpn60/GroEL/TCP-1 family
Similarity search - Domain/homology
T-complex protein 1 subunit epsilon
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM
AuthorsDarrow MC / Sergeeva OA / Isas JM / Galaz-Montoya J / King JA / Langen R / Schmid MF / Chiu W
CitationJournal: J Biol Chem / Year: 2014
Title: Biochemical characterization of mutants in chaperonin proteins CCT4 and CCT5 associated with hereditary sensory neuropathy.
Authors: Oksana A Sergeeva / Meme T Tran / Cameron Haase-Pettingell / Jonathan A King /
Abstract: Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the ...Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.
History
DepositionMar 25, 2015-
Header (metadata) releaseApr 15, 2015-
Map releaseJun 3, 2015-
UpdateJul 22, 2015-
Current statusJul 22, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2962.tif

Downloads & links

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Map

FileDownload / File: emd_2962.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle tomography reconstruction of unoccupied CCT5 complex after incubation with mutant huntington.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.7 Å/pix.
x 72 pix.
= 338.688 Å		4.7 Å/pix.
x 72 pix.
= 338.688 Å		4.7 Å/pix.
x 72 pix.
= 338.688 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.704 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0781 / Movie #1: 0.1
Minimum - Maximum-0.16106577 - 0.35770315
Average (Standard dev.)0.00772435 (±0.06003072)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 338.688 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.7044.7044.704
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z338.688338.688338.688
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-0.1610.3580.008

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Supplemental data

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Sample components

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Entire : Unoccupied CCT5 complex after incubation with mutant huntingtin e...

EntireName: Unoccupied CCT5 complex after incubation with mutant huntingtin exon 1.
Components
  • Sample: Unoccupied CCT5 complex after incubation with mutant huntingtin exon 1.
  • Protein or peptide: chaperonin containing TCP1, subunit 5 (epsilon) complex

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Supramolecule #1000: Unoccupied CCT5 complex after incubation with mutant huntingtin e...

SupramoleculeName: Unoccupied CCT5 complex after incubation with mutant huntingtin exon 1.
type: sample / ID: 1000
Oligomeric state: 16 CCT5 subunits make up one CCT5 complex.
Number unique components: 1

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

MacromoleculeName: chaperonin containing TCP1, subunit 5 (epsilon) complex
type: protein_or_peptide / ID: 1 / Name.synonym: CCT5 Complex / Oligomeric state: Hexadecamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasm
Molecular weightExperimental: 1.0 MDa / Theoretical: 960 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: RIL / Recombinant plasmid: pET21b
SequenceUniProtKB: T-complex protein 1 subunit epsilon / GO: chaperonin-containing T-complex, protein folding / InterPro: Chaperonin Cpn60/GroEL/TCP-1 family

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 8 / Details: 20 mM Tris, 150 mM NaCl, 1 mM DTT, 1% glycerol
GridDetails: 200 mesh Quantifoil copper grid R 2/2 holey carbon support, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK III
Method: 1x or 2x double sided blotting for 1 or 2 seconds each blot before plunging.

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Electron microscopy #1

Microscopy ID1
MicroscopeJEOL 2200FS
TemperatureAverage: 95 K
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
DateFeb 12, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 2.0 e/Å2 / Camera length: 1200
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 25000
Sample stageSpecimen holder: LN2 cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -55 ° / Tilt series - Axis1 - Max angle: 55 °

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Electron microscopy #2

Microscopy ID2
MicroscopeJEOL 2200FS
TemperatureAverage: 95 K
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
DateOct 31, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 2.0 e/Å2 / Camera length: 1200
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 25000
Sample stageSpecimen holder: LN2 cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -55 ° / Tilt series - Axis1 - Max angle: 55 °

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Image processing

DetailsSubvolumes were manually selected using IMOD for coordinates and EMAN2 for boxing. Further processing was completed in EMAN2.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Software - Name: IMOD, EMAN2 / Number subtomograms used: 300

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