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- PDB-8sh0: Structure of human POT1 DNA binding domain bound to a 5'-phosphor... -

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Basic information

Entry
Database: PDB / ID: 8sh0
TitleStructure of human POT1 DNA binding domain bound to a 5'-phosphorylated junction of a telomeric DNA hairpin with a 3'-overhang
Components
  • DNA (5'-D(P*CP*CP*AP*GP*CP*AP*GP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
  • Protection of telomeres protein 1
KeywordsDNA BINDING PROTEIN/DNA / shelterin / telomere / DNA junction / 5'-phosphorylated / POT-hole / chromosome end protection / POT1 / DBD / POT1-DNA complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


positive regulation of DNA strand elongation / regulation of DNA helicase activity / positive regulation of DNA helicase activity / G-rich single-stranded DNA binding / telomere assembly / 8-hydroxy-2'-deoxyguanosine DNA binding / telomeric D-loop binding / regulation of double-strand break repair via nonhomologous end joining / positive regulation of helicase activity / telomerase inhibitor activity ...positive regulation of DNA strand elongation / regulation of DNA helicase activity / positive regulation of DNA helicase activity / G-rich single-stranded DNA binding / telomere assembly / 8-hydroxy-2'-deoxyguanosine DNA binding / telomeric D-loop binding / regulation of double-strand break repair via nonhomologous end joining / positive regulation of helicase activity / telomerase inhibitor activity / DEAD/H-box RNA helicase binding / establishment of protein localization to telomere / telomeric D-loop disassembly / shelterin complex / Telomere C-strand synthesis initiation / regulation of telomere maintenance via telomerase / Telomere C-strand (Lagging Strand) Synthesis / : / positive regulation of telomere maintenance / nuclear telomere cap complex / single-stranded telomeric DNA binding / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / G-rich strand telomeric DNA binding / telomere capping / DNA duplex unwinding / telomeric DNA binding / negative regulation of telomere maintenance via telomerase / Telomere Extension By Telomerase / telomere maintenance via telomerase / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / : / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / positive regulation of telomere maintenance via telomerase / Inhibition of DNA recombination at telomere / Meiotic synapsis / DNA Damage/Telomere Stress Induced Senescence / chromosome, telomeric region / nucleoplasm
Similarity search - Function
: / Protection of telomeres protein 1 (POT1), C-terminal insertion domain / Protection of telomeres protein 1, ssDNA-binding domain / ssDNA-binding domain of telomere protection protein / Protection of telomeres protein 1 / Telomeric single stranded DNA binding POT1/Cdc13 / Telomeric single stranded DNA binding POT1/CDC13 / Telomeric single stranded DNA binding POT1/CDC13 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
ACETATE ION / DNA / DNA (> 10) / Protection of telomeres protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.16 Å
AuthorsNandakumar, J. / Tesmer, V.M.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120094 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM148276 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)R01HD108809 United States
American Cancer SocietyRSG-17-037-01-DMC United States
CitationJournal: Science / Year: 2023
Title: Human POT1 protects the telomeric ds-ss DNA junction by capping the 5' end of the chromosome.
Authors: Tesmer, V.M. / Brenner, K.A. / Nandakumar, J.
History
DepositionApr 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protection of telomeres protein 1
B: DNA (5'-D(P*CP*CP*AP*GP*CP*AP*GP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,2445
Polymers40,0672
Non-polymers1773
Water2,162120
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3280 Å2
ΔGint-14 kcal/mol
Surface area17450 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.230, 67.230, 143.373
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z
Components on special symmetry positions
IDModelComponents
11B-101-

HOH

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Components

#1: Protein Protection of telomeres protein 1 / hPot1 / POT1-like telomere end-binding protein


Mass: 33811.828 Da / Num. of mol.: 1 / Fragment: DNA binding domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POT1 / Plasmid: pFBHTb-Smt3star-hPOT1-1-299
Details (production host): human POT1 aa 1-299 cloned downstream of a sumo-star tag in the pFastBac vector with an N-terminal His tag
Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NUX5
#2: DNA chain DNA (5'-D(P*CP*CP*AP*GP*CP*AP*GP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')


Mass: 6255.040 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H3O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 120 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.33 Å3/Da / Density % sol: 47.31 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / pH: 8
Details: hDBD complex with 5prime-p-hp-ss1-12 was crystallized at 16C by the hanging drop method in a drop containing 0.8 microliter of 13 mg/ml protein-DNA complex (in 25 mM Tris (pH 8), 100 mM ...Details: hDBD complex with 5prime-p-hp-ss1-12 was crystallized at 16C by the hanging drop method in a drop containing 0.8 microliter of 13 mg/ml protein-DNA complex (in 25 mM Tris (pH 8), 100 mM NaCl, and 2 mM DTT) and 0.8 microliter well solution (0.1 M NaOAc (pH 5.2) and 10% PEG 20,000). Crystals were harvested in 25 mM Tris (pH 8), 100 mM NaCl, 0.1 M NaOAc (pH 5.2), and 10% PEG 20,000 and cryoprotected in harvesting solution supplemented with 30% ethylene glycol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1.12723 Å
DetectorType: DECTRIS EIGER2 X 9M / Detector: PIXEL / Date: Jul 28, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.12723 Å / Relative weight: 1
ReflectionResolution: 2.16→71.69 Å / Num. obs: 20897 / % possible obs: 100 % / Redundancy: 19.3 % / CC1/2: 0.999 / Rmerge(I) obs: 0.086 / Net I/σ(I): 19.6
Reflection shellResolution: 2.16→2.23 Å / Rmerge(I) obs: 1.131 / Num. unique obs: 36315 / CC1/2: 0.858

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.16→45.19 Å / SU ML: 0.1923 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.272
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2214 1745 4.48 %
Rwork0.18 37199 -
obs0.1818 20846 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 63.64 Å2
Refinement stepCycle: LAST / Resolution: 2.16→45.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2339 420 12 120 2891
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00342874
X-RAY DIFFRACTIONf_angle_d0.64613989
X-RAY DIFFRACTIONf_chiral_restr0.0443455
X-RAY DIFFRACTIONf_plane_restr0.0043430
X-RAY DIFFRACTIONf_dihedral_angle_d18.90071065
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkRefine-ID% reflection obs (%)
2.16-2.220.28981660.2492X-RAY DIFFRACTION99.97
2.22-2.290.26941360.2293X-RAY DIFFRACTION100
2.29-2.380.26071280.2175X-RAY DIFFRACTION100
2.38-2.470.26161520.2527X-RAY DIFFRACTION99.97
2.47-2.580.31471460.2298X-RAY DIFFRACTION100
2.58-2.720.24691550.2032X-RAY DIFFRACTION99.91
2.72-2.890.2972060.2476X-RAY DIFFRACTION99.88
2.89-3.110.26211500.2207X-RAY DIFFRACTION100
3.11-3.430.22661000.1807X-RAY DIFFRACTION99.97
3.43-3.920.20231460.1652X-RAY DIFFRACTION99.97
3.92-4.940.17381300.1413X-RAY DIFFRACTION100
4.94-45.190.18731300.1562X-RAY DIFFRACTION99.91
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.27499705727-0.173399153837-0.3728638147483.594121180850.7019245229143.270035482570.050870999788-0.3744178932350.1958474352180.4901162693060.01158521375790.138417121677-0.3417452268510.194697044207-0.06898306383950.601934094101-0.03560513250490.01622110296070.2931851684940.03684474269250.303963747694-35.350075072219.684727498718.3831127361
24.284957654140.336866343078-0.9054487254294.151747587320.6683366222994.69639567628-0.0777320615252-0.437481296487-0.1478190255320.3347234506420.118650678848-0.5793307019370.01950542711180.570828457154-0.0316526645580.3963330604510.00997085334442-0.02250639720620.3865266333660.07433954225860.473081032418-17.34901093854.267196321656.70798622131
30.92433311554-0.268882267593-0.6353682329933.314166875240.6663820516453.94359613172-0.0989896645140.506199103054-0.198402829517-0.347284712090.0689292427101-0.7890294360650.2020397122010.372081149894-0.1192067048380.352520512526-0.03456399879280.05414221538740.3372894515010.03333443981640.46658211283-15.99377897491.45001077613-3.48168162653
40.6718785393160.443196878409-0.1246635435240.280894878207-0.0641725232860.423527847073-0.1799332546280.0726078458778-0.49319622976-0.5671112930510.000607319694018-1.083208823690.6934456672640.2135161931070.176049060970.7061196710610.0399057213970.1296398509150.3896042964930.1324978081170.794183565905-14.478979801-3.01221313633.83700173362
54.088593154360.429602362427-2.117205446092.148373845650.1311854834172.23738049270.013257565088-0.4359147146970.07585795302550.2647116792710.0308745168761-0.362530462528-0.06051030368660.407635088805-0.004313078993450.425807057624-0.045610939766-0.05893542092720.3887535991490.09120095105460.444459340887-16.60920667449.790584332845.11929465079
66.42345957935-2.960185777890.6138825634031.756357971220.3444200792281.94894626432-0.1700134949210.555695654091-0.7859671664360.511086200344-0.2283773099040.901870200519-0.466579130564-0.6901507838450.3051666566970.9230155612870.170764823906-0.00830390976350.677654461386-0.009664847211760.870731007586-52.964440395925.52115288548.34194795792
70.977713064910.622263999388-1.01748340820.9109663741360.3510859941423.67827544021-0.651513373679-0.247430648644-0.8332041181510.4558811453840.04207862024950.1464859175230.5989747198830.5986939906020.5018737134440.6874867819330.03699234852880.1827783386970.4180232320230.1720638310610.584024961072-36.0245271389-0.73371848177213.4242021759
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 6 through 143 )AA6 - 1431 - 138
22chain 'A' and (resid 144 through 184 )AA144 - 184139 - 179
33chain 'A' and (resid 185 through 232 )AA185 - 232180 - 227
44chain 'A' and (resid 233 through 258 )AA233 - 258228 - 253
55chain 'A' and (resid 259 through 299 )AA259 - 299254 - 294
66chain 'B' and (resid -7 through 2 )BB-7 - 2
77chain 'B' and (resid 3 through 12 )BB3 - 12

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