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- PDB-5dfn: Structure of Tetrahymena Telomerase P45 C-terminal domain -

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Basic information

Entry
Database: PDB / ID: 5dfn
TitleStructure of Tetrahymena Telomerase P45 C-terminal domain
ComponentsTelomerase associated protein p45
KeywordsNUCLEAR PROTEIN / Telomerase / P45 / CST complex / Stn1 / Winged Helix / WH domain / WHtH / Winged Helix turn Helix
Function / homologytelomerase holoenzyme complex / telomere maintenance via telomerase / chromosome, telomeric region / metal ion binding / Telomerase-associated protein of 45 kDa / Telomerase-associated protein of 45 kDa
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.382 Å
Model detailsTelomerase protein P45C
AuthorsChan, H. / Cascio, D. / Sawaya, M.R. / 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, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 28, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2015Group: Database references
Revision 1.2Mar 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_struct_oper_list / struct_ncs_dom_lim
Item: _citation.journal_id_CSD / _database_2.pdbx_DOI ..._citation.journal_id_CSD / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.symmetry_operation / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Telomerase associated protein p45
B: Telomerase associated protein p45


Theoretical massNumber of molelcules
Total (without water)50,4212
Polymers50,4212
Non-polymers00
Water1086
1
A: Telomerase associated protein p45


Theoretical massNumber of molelcules
Total (without water)25,2111
Polymers25,2111
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Telomerase associated protein p45


Theoretical massNumber of molelcules
Total (without water)25,2111
Polymers25,2111
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Telomerase associated protein p45

A: Telomerase associated protein p45

A: Telomerase associated protein p45

A: Telomerase associated protein p45


Theoretical massNumber of molelcules
Total (without water)100,8434
Polymers100,8434
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_755-x+2,-y,z1
crystal symmetry operation3_757-x+2,y,-z+21
crystal symmetry operation4_557x,-y,-z+21
Buried area10210 Å2
ΔGint-43 kcal/mol
Surface area31040 Å2
MethodPISA
4
B: Telomerase associated protein p45

B: Telomerase associated protein p45

B: Telomerase associated protein p45

B: Telomerase associated protein p45


Theoretical massNumber of molelcules
Total (without water)100,8434
Polymers100,8434
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_556x,-y+1/2,-z+3/21
crystal symmetry operation11_656-x+3/2,y,-z+3/21
crystal symmetry operation14_655-x+3/2,-y+1/2,z1
Buried area11880 Å2
ΔGint-49 kcal/mol
Surface area31860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)88.030, 124.620, 173.770
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number22
Space group name H-MF222
Components on special symmetry positions
IDModelComponents
11A-303-

HOH

21B-301-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: THR / Beg label comp-ID: THR

Dom-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1GLNGLNchain AAA18 - 20619 - 207
2SERSERchain BBB18 - 21319 - 214

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Components

#1: Protein Telomerase associated protein p45


Mass: 25210.664 Da / Num. of mol.: 2 / Fragment: C-terminal domain (UNP residues 161-373)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote) / Gene: P45 / Plasmid: pETDuet / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q237B3, UniProt: Q6JXI5*PLUS
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.36 Å3/Da / Density % sol: 47.95 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 14 mg/ml P45 protein, 0.1 M Na-HEPES buffer, 5%v/v MPD, 10%w/v PEG-6000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 16, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 2.38→86.89 Å / Num. obs: 18884 / % possible obs: 97.8 % / Observed criterion σ(I): -3 / Redundancy: 5.35 % / Biso Wilson estimate: 53.1 Å2 / Rmerge F obs: 0.999 / Rmerge(I) obs: 0.064 / Rrim(I) all: 0.072 / Χ2: 1.034 / Net I/σ(I): 14.07 / Num. measured all: 101012
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Rmerge F obsRmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsRrim(I) all% possible all
2.38-2.440.8670.7282.545429140911070.81478.6
2.44-2.510.9270.5363.597312137313680.59699.6
2.51-2.580.9410.473.966602132313130.52699.2
2.58-2.660.9680.3425.587436130913050.37899.7
2.66-2.750.9760.2936.317091126412570.32399.4
2.75-2.850.9840.2377.636765121012070.26299.8
2.85-2.950.990.28.66553119011870.22199.7
2.95-3.080.9920.15210.416047114011360.1799.6
3.08-3.210.9910.12312.225464108210700.13898.9
3.21-3.370.9970.09215.585908105210450.10299.3
3.37-3.550.9960.07319.7155629979890.08199.2
3.55-3.770.9970.06520.9450319449380.07299.4
3.77-4.030.9970.05424.4545458878800.06199.2
4.03-4.350.9970.04826.7341798168070.05398.9
4.35-4.760.9980.04428.8642507847810.04999.6
4.76-5.330.9980.04329.6737806946930.04899.9
5.33-6.150.9980.04625.0529716206100.05198.4
6.15-7.530.9980.04228.328925395360.04699.4
7.53-10.650.9980.03532.0621044204160.03999
10.65-86.890.9990.02832.7210912502390.03195.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
XSCALEdata scaling
PHASER2.5.6phasing
PHENIX(phenix.refine: 1.9_1692)refinement
PDB_EXTRACT3.15data extraction
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.382→86.885 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 33.97 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2643 1883 10 %
Rwork0.207 16951 -
obs0.2127 18834 97.59 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 161.49 Å2 / Biso mean: 62.762 Å2 / Biso min: 29.84 Å2
Refinement stepCycle: final / Resolution: 2.382→86.885 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2730 0 0 6 2736
Biso mean---52.01 -
Num. residues----339
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0082779
X-RAY DIFFRACTIONf_angle_d1.0093763
X-RAY DIFFRACTIONf_chiral_restr0.041435
X-RAY DIFFRACTIONf_plane_restr0.005469
X-RAY DIFFRACTIONf_dihedral_angle_d15.0741007
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A1509X-RAY DIFFRACTION6.599TORSIONAL
12B1509X-RAY DIFFRACTION6.599TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.3825-2.44690.41511160.32871035115179
2.4469-2.51890.33291470.27471324147199
2.5189-2.60020.33811430.25181303144699
2.6002-2.69310.28171420.24181288143099
2.6931-2.8010.28061460.23121314146099
2.801-2.92840.36811480.24071340148899
2.9284-3.08280.35461460.24791305145199
3.0828-3.2760.32091470.2451322146999
3.276-3.5290.27031450.2221308145399
3.529-3.88410.29081490.2091335148499
3.8841-4.44610.22771490.17041342149199
4.4461-5.60150.19991490.176113411490100
5.6015-86.94280.2291560.18991394155098
Refinement TLS params.Method: refined / Origin x: 76.4309 Å / Origin y: 15.5674 Å / Origin z: 148.336 Å
111213212223313233
T0.554 Å20.0189 Å2-0.0192 Å2-0.3236 Å20.0298 Å2--0.477 Å2
L0.3221 °2-0.0377 °20.0205 °2-0.539 °20.0566 °2--0.5161 °2
S-0.0372 Å °0.117 Å °-0.0204 Å °-0.3229 Å °0.0164 Å °0.0411 Å °-0.0071 Å °0.019 Å °0.0111 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA18 - 206
2X-RAY DIFFRACTION1allB18 - 213
3X-RAY DIFFRACTION1allC1 - 10

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