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- EMDB-12957: Cryo-EM structure of the human R2TP-TTT complex -

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Basic information

Entry
Database: EMDB / ID: EMD-12957
TitleCryo-EM structure of the human R2TP-TTT complex
Map dataCryo-EM structure of the human R2TP-TTT complex
Sample
  • Complex: RUVBL1-RUVBL2-RPAP3-PIH1D1 in complex with TELO2-TTI1-TTI2 adaptor proteins
Function / homology
Function and homology information


positive regulation of DNA damage checkpoint / 'de novo' cotranslational protein folding / TTT Hsp90 cochaperone complex / promoter-enhancer loop anchoring activity / telomerase RNA localization to Cajal body / regulation of DNA strand elongation / positive regulation of telomere maintenance in response to DNA damage / TORC2 complex / establishment of protein localization to chromatin / TORC1 complex ...positive regulation of DNA damage checkpoint / 'de novo' cotranslational protein folding / TTT Hsp90 cochaperone complex / promoter-enhancer loop anchoring activity / telomerase RNA localization to Cajal body / regulation of DNA strand elongation / positive regulation of telomere maintenance in response to DNA damage / TORC2 complex / establishment of protein localization to chromatin / TORC1 complex / R2TP complex / regulation of TOR signaling / dynein axonemal particle / Swr1 complex / RPAP3/R2TP/prefoldin-like complex / Ino80 complex / regulation of double-strand break repair / box C/D snoRNP assembly / protein folding chaperone complex / telomeric DNA binding / regulation of chromosome organization / NuA4 histone acetyltransferase complex / regulation of DNA replication / TFIID-class transcription factor complex binding / regulation of embryonic development / MLL1 complex / Telomere Extension By Telomerase / positive regulation of double-strand break repair via homologous recombination / regulation of DNA repair / RNA polymerase II core promoter sequence-specific DNA binding / DNA helicase activity / Deposition of new CENPA-containing nucleosomes at the centromere / TBP-class protein binding / telomere maintenance / positive regulation of DNA repair / cellular response to estradiol stimulus / Hsp90 protein binding / Formation of the beta-catenin:TCF transactivating complex / negative regulation of canonical Wnt signaling pathway / euchromatin / DNA Damage Recognition in GG-NER / beta-catenin binding / ADP binding / chromatin DNA binding / nuclear matrix / transcription corepressor activity / cellular response to UV / UCH proteinases / positive regulation of canonical Wnt signaling pathway / nucleosome / unfolded protein binding / protein folding / HATs acetylate histones / ATPase binding / regulation of apoptotic process / DNA recombination / spermatogenesis / molecular adaptor activity / DNA helicase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / transcription coactivator activity / chromosome, telomeric region / regulation of cell cycle / protein stabilization / Ub-specific processing proteases / nuclear speck / nuclear body / ciliary basal body / cadherin binding / chromatin remodeling / RNA polymerase II cis-regulatory region sequence-specific DNA binding / ribonucleoprotein complex / cell division / DNA repair / centrosome / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / protein-containing complex binding / protein kinase binding / positive regulation of DNA-templated transcription / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Tti2 family / Tti2 family / TEL2-interacting protein 1 / : / : / TELO2-interacting protein 1 / TTI1 N-terminal TPR domain / TELO2-interacting protein 1 homologue second TPR domain / TTI1 C-terminal TPR domain / Telomere length regulation protein, conserved domain ...Tti2 family / Tti2 family / TEL2-interacting protein 1 / : / : / TELO2-interacting protein 1 / TTI1 N-terminal TPR domain / TELO2-interacting protein 1 homologue second TPR domain / TTI1 C-terminal TPR domain / Telomere length regulation protein, conserved domain / TEL2, C-terminal domain superfamily / : / Telomere length regulation protein / RuvB-like / RuvB-like, AAA-lid domain / RuvBL1/2, DNA/RNA binding domain / TIP49 P-loop domain / TIP49 AAA-lid domain / TIP49, P-loop domain / Armadillo-like helical / Armadillo-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
TELO2-interacting protein 1 homolog / TELO2-interacting protein 2 / RuvB-like 2 / RuvB-like 1 / Telomere length regulation protein TEL2 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsPal M / Llorca O / Pearl L
Funding support United Kingdom, Spain, 4 items
OrganizationGrant numberCountry
Wellcome Trust095605/Z/11/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R01678X/1 United Kingdom
European Regional Development FundSAF2017-82632-P Spain
European Regional Development Fund Spain
CitationJournal: Cell Rep / Year: 2021
Title: Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.
Authors: Mohinder Pal / Hugo Muñoz-Hernandez / Dennis Bjorklund / Lihong Zhou / Gianluca Degliesposti / J Mark Skehel / Emma L Hesketh / Rebecca F Thompson / Laurence H Pearl / Oscar Llorca / Chrisostomos Prodromou /
Abstract: The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA ...The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.
History
DepositionMay 17, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0048
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0048
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12957.png

Downloads & links

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Map

FileDownload / File: emd_12957.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the human R2TP-TTT complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 280 pix.
= 293.44 Å		1.05 Å/pix.
x 280 pix.
= 293.44 Å		1.05 Å/pix.
x 280 pix.
= 293.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0048 / Movie #1: 0.0048
Minimum - Maximum-0.009024014 - 0.031790406
Average (Standard dev.)0.00042016292 (±0.0017494085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 293.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0481.0481.048
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z293.440293.440293.440
α/β/γ90.00090.00090.000
start NX/NY/NZ29290
NX/NY/NZ140137200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0090.0320.000

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Supplemental data

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Half map: Cryo-EM structure of the human R2TP-TTT complex (half maps)

Fileemd_12957_half_map_1.map
AnnotationCryo-EM structure of the human R2TP-TTT complex (half maps)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of the human R2TP-TTT complex (half maps)

Fileemd_12957_half_map_2.map
AnnotationCryo-EM structure of the human R2TP-TTT complex (half maps)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RUVBL1-RUVBL2-RPAP3-PIH1D1 in complex with TELO2-TTI1-TTI2 adapto...

EntireName: RUVBL1-RUVBL2-RPAP3-PIH1D1 in complex with TELO2-TTI1-TTI2 adaptor proteins
Components
  • Complex: RUVBL1-RUVBL2-RPAP3-PIH1D1 in complex with TELO2-TTI1-TTI2 adaptor proteins

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Supramolecule #1: RUVBL1-RUVBL2-RPAP3-PIH1D1 in complex with TELO2-TTI1-TTI2 adapto...

SupramoleculeName: RUVBL1-RUVBL2-RPAP3-PIH1D1 in complex with TELO2-TTI1-TTI2 adaptor proteins
type: complex / ID: 1 / Parent: 0
Details: The complex was reconstituted using purified proteins from E.coli and Sf9 cells
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 800 kDa/nm

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
140.0 mMNaClSodium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 287.15 K / Instrument: LEICA PLUNGER / Details: 3Sec blotting time.
DetailsThis sample was gel filtered

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 10 / Number real images: 2400 / Average exposure time: 9.0 sec. / Average electron dose: 60.17 e/Å2 / Details: Data was collected in movie mode with 40 fractions
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 130000
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: 168882
CTF correctionSoftware - Name: RELION (ver. 3.0) / Software - details: CTFFIND
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Software - details: RELION / Number images used: 90575
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0) / Software - details: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0) / Software - details: RELION
Final 3D classificationNumber classes: 3 / Avg.num./class: 44 / Software - Name: RELION (ver. 3.1) / Software - details: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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