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- PDB-8sh1: Structure of human POT1 DNA binding domain bound to a 5'-phosphor... -

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Basic information

Entry
Database: PDB / ID: 8sh1
TitleStructure of human POT1 DNA binding domain bound to a 5'-phosphorylated junction of a telomeric double-stranded DNA duplex with a 3'-overhang
Components
  • DNA (5'-D(*CP*GP*CP*GP*CP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
  • DNA (5'-D(P*CP*TP*AP*AP*CP*GP*CP*GP*CP*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
DNA / DNA (> 10) / Protection of telomeres protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 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(*CP*GP*CP*GP*CP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
C: DNA (5'-D(P*CP*TP*AP*AP*CP*GP*CP*GP*CP*G)-3')


Theoretical massNumber of molelcules
Total (without water)43,7213
Polymers43,7213
Non-polymers00
Water81145
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3810 Å2
ΔGint-19 kcal/mol
Surface area19220 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.470, 170.620, 173.960
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number23
Space group name H-MI222
Space group name HallI22
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z
#4: -x,-y,z
#5: x+1/2,y+1/2,z+1/2
#6: x+1/2,-y+1/2,-z+1/2
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2

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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(*CP*GP*CP*GP*CP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')


Mass: 6879.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (5'-D(P*CP*TP*AP*AP*CP*GP*CP*GP*CP*G)-3')


Mass: 3029.993 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 45 / 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: 4.96 Å3/Da / Density % sol: 75.21 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 8
Details: hDBD complex with 5prime-P-ds-ss1-12 was crystallized at 16C by the sitting drop method in a drop containing 0.5 microliter 14 mg/ml protein-DNA complex (in 25 mM Tris (pH 8), 100 mM NaCl, ...Details: hDBD complex with 5prime-P-ds-ss1-12 was crystallized at 16C by the sitting drop method in a drop containing 0.5 microliter 14 mg/ml protein-DNA complex (in 25 mM Tris (pH 8), 100 mM NaCl, and 2 mM DTT) and 0.5 microliter well solution (0.02 M MgCl2, 0.9 M Hepes (pH 7.5), and 20% polyacrylic acid N100 sodium salt, derived from condition G2 of the JCSG+ screen (NeXtal). Crystals were harvested in 25 mM Tris (pH 8), 100 mM NaCl, 0.02 M MgCl2, 0.1 M Hepes (pH 7.5), and 22% polyacrylic acid N100 sodium salt and cryoprotected in harvesting solution supplemented with 35% 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: 1.12713 Å
DetectorType: DECTRIS EIGER2 X 9M / Detector: PIXEL / Date: Jun 30, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.12713 Å / Relative weight: 1
ReflectionResolution: 2.6→86.98 Å / Num. obs: 27182 / % possible obs: 99.7 % / Redundancy: 13.4 % / CC1/2: 0.997 / Rmerge(I) obs: 0.146 / Net I/σ(I): 10.2
Reflection shellResolution: 2.6→2.72 Å / Num. unique obs: 45826 / CC1/2: 0.27

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→42.66 Å / SU ML: 0.6423 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 39.3949
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2544 2537 4.98 %
Rwork0.2303 48380 -
obs0.2315 27070 98.5 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 128.89 Å2
Refinement stepCycle: LAST / Resolution: 2.6→42.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2333 664 0 45 3042
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00283129
X-RAY DIFFRACTIONf_angle_d0.59034386
X-RAY DIFFRACTIONf_chiral_restr0.04501
X-RAY DIFFRACTIONf_plane_restr0.0033437
X-RAY DIFFRACTIONf_dihedral_angle_d21.52721179
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.650.5671450.5509X-RAY DIFFRACTION91.21
2.65-2.710.49031520.5195X-RAY DIFFRACTION94.15
2.71-2.760.48531320.4999X-RAY DIFFRACTION98.21
2.76-2.830.49441330.4689X-RAY DIFFRACTION99.3
2.83-2.90.47061530.4263X-RAY DIFFRACTION99.38
2.9-2.980.39081450.3892X-RAY DIFFRACTION99.62
2.98-3.060.39911490.3491X-RAY DIFFRACTION99
3.06-3.160.28961150.3074X-RAY DIFFRACTION98.9
3.16-3.280.35491320.3016X-RAY DIFFRACTION99.55
3.28-3.410.28961460.2698X-RAY DIFFRACTION99.3
3.41-3.560.25891160.2341X-RAY DIFFRACTION97.44
3.56-3.750.28181460.2247X-RAY DIFFRACTION99.89
3.75-3.990.24491580.2224X-RAY DIFFRACTION99.86
3.99-4.290.22261320.217X-RAY DIFFRACTION99.79
4.29-4.720.20521700.1756X-RAY DIFFRACTION99.93
4.73-5.410.21741400.1863X-RAY DIFFRACTION100
5.41-6.810.20261110.2161X-RAY DIFFRACTION98.74
6.81-42.660.21241620.17350X-RAY DIFFRACTION98.79
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)
14.93756726715-0.23674890625-0.5253922807383.67651315406-1.414996940865.049780499090.375444351432-0.620175865531-0.6306510809290.367687763636-0.039927898462-0.6312190598750.7066502989530.180330064895-0.4326893984011.19537356797-0.0495128684058-0.3045133730840.9993315477880.14891283191.038740923530.290478313749-15.0660400611-52.7138105637
24.894690840240.036475067144-0.1448939525213.716787484570.4070326555764.976607588740.304850564608-1.15562037955-0.588872654191.118275400280.386984812660.1041906505061.22638518482-0.710464888941-0.5404329049011.56694683125-0.173862100595-0.2799624314041.245045824270.3592768286691.10705010158-6.81705700539-17.5889362224-46.9687905592
37.28887530081-1.139998809371.975673504135.68534940142-2.338443119164.435492345070.575451929344-0.426661348006-0.5184441309490.345166932376-0.1861053127730.2869479791650.121264826409-0.331090416583-0.2545169791970.750419527546-0.123182022946-0.1228862691550.666387103010.1056158598220.742124921689-17.6754960414-12.911859612-71.896219684
44.26992032454-0.3691267367092.464489874683.151033070550.3432225896494.043638634350.665577822863-0.0149590717139-0.9893004591720.12330984372-0.2223144662860.2249158372971.07334921809-0.304318730449-0.3560498145780.817109154668-0.0634643252567-0.1043255166240.7824884499610.04443891831950.930823281226-19.2860824171-16.7085517895-75.9807988058
51.659833824950.473597851928-1.555172927720.6730612150930.5426740376953.48768874233-0.4308211803920.5717980957520.5210214230790.538331392238-0.96398157292-0.6323799423860.2190756345250.3424479482971.068109515782.874621556260.579500842834-0.3451948000361.831334211310.8606247437593.2264793775811.3420420554-41.7575646237-42.6880043677
62.114174541920.4227082695531.05345585152.072027176142.720852916283.60632151947-0.1344330013930.2721260753420.2681855611050.0100373150059-0.0742028549573-0.2085430936510.2500397197570.7816671185090.1750382453760.921476235306-0.0350258033329-0.09391953470141.131487688930.2476833581671.459096952164.50092858405-11.1913324621-67.5137691592
72.8729073943-1.71368197046-0.0218208391981.05113062970.04720822652221.25848781314-0.06289067175550.654332718178-0.2822220429730.159529795297-1.22769701899-1.843320766680.9289351692471.154756162270.9354277791763.223811593250.427147154301-0.7498528538021.532095978640.7339627649662.8396417404711.9247694315-41.5611709226-43.0772387592
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 89 )AB6 - 891 - 84
22chain 'A' and (resid 90 through 143 )AB90 - 14385 - 138
33chain 'A' and (resid 144 through 184 )AB144 - 184139 - 179
44chain 'A' and (resid 185 through 298 )AB185 - 298180 - 293
55chain 'B' and (resid -9 through 0 )BC-9 - 0
66chain 'B' and (resid 1 through 12 )BC1 - 12
77chain 'C' and (resid 0 through 9 )CD0 - 9

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