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- EMDB-6443: CryoEM structure of endogenously assembled Tetrahymena telomerase... -

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Basic information

Entry
Database: EMDB / ID: EMD-6443
TitleCryoEM structure of endogenously assembled Tetrahymena telomerase holoenzyme at 8.9 Angstrom resolution
Map dataTetrahymena telomerase holoenzyme, map refined using a soft-edge mask that excludes the flexible p75-p45-p19 subcomplex
Sample
  • Sample: Telomerase holoenzyme from Tetrahymena thermophila
  • Protein or peptide: Telomerase holoenzyme
Keywordstelomerase / telomere / Tetrahymena
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.9 Å
AuthorsJiang J / Chan H / Cash DD / Miracco EJ / Loo RRO / Upton HE / Cascio D / Johnson RO / Collins K / Loo JA ...Jiang J / Chan H / Cash DD / Miracco EJ / Loo RRO / Upton HE / Cascio D / Johnson RO / Collins K / Loo JA / Zhou ZH / Feigon J
CitationJournal: Science / Year: 2015
Title: Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions.
Authors: Jiansen Jiang / Henry Chan / Darian D Cash / Edward J Miracco / Rachel R Ogorzalek Loo / Heather E Upton / Duilio Cascio / Reid O'Brien Johnson / Kathleen Collins / Joseph A Loo / Z Hong Zhou / Juli Feigon /
Abstract: Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3'-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the ...Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3'-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the cryo-electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric replication protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunit interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Our findings provide structural and mechanistic insights into telomerase holoenzyme function.
History
DepositionAug 27, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseOct 28, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6443.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTetrahymena telomerase holoenzyme, map refined using a soft-edge mask that excludes the flexible p75-p45-p19 subcomplex
Voxel sizeX=Y=Z: 2.72 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.05309743 - 0.27401197
Average (Standard dev.)0.0004303 (±0.01568625)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 348.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.722.722.72
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z348.160348.160348.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0530.2740.000

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Supplemental data

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Sample components

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Entire : Telomerase holoenzyme from Tetrahymena thermophila

EntireName: Telomerase holoenzyme from Tetrahymena thermophila
Components
  • Sample: Telomerase holoenzyme from Tetrahymena thermophila
  • Protein or peptide: Telomerase holoenzyme

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Supramolecule #1000: Telomerase holoenzyme from Tetrahymena thermophila

SupramoleculeName: Telomerase holoenzyme from Tetrahymena thermophila / type: sample / ID: 1000 / Oligomeric state: heterodecamer / Number unique components: 1
Molecular weightTheoretical: 560 KDa

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Macromolecule #1: Telomerase holoenzyme

MacromoleculeName: Telomerase holoenzyme / type: protein_or_peptide / ID: 1 / Name.synonym: Telomerase / Number of copies: 1 / Oligomeric state: heterodecamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / synonym: Tetrahymena / Organelle: nucleus
Molecular weightTheoretical: 560 KDa

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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

BufferpH: 8
Details: 20 mM HEPES-NaOH, 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP-HCl
GridDetails: 300 mesh Quantifoil R2/1 holey grids, glow-discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 36764 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder: Liquid nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJan 1, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 4210 / Average electron dose: 30 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 40754
DetailsThe refinement was performed using RELION.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

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SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 4

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 5

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 6

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 7

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 8

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 9

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 10

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 11

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 12

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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