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- PDB-8uzt: Mitochondrial single-stranded binding protein bound to DNA -

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Basic information

Entry
Database: PDB / ID: 8uzt
TitleMitochondrial single-stranded binding protein bound to DNA
Components
  • Single-stranded DNA-binding protein, mitochondrial
  • dTDNA
KeywordsDNA BINDING PROTEIN/DNA / single-stranded binding protein / DNA BINDING PROTEIN / mitochondrial SSB / SSB / mitochondrial replication protein / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


positive regulation of mitochondrial DNA replication / positive regulation of helicase activity / mitochondrial DNA replication / DNA unwinding involved in DNA replication / mitochondrial nucleoid / Mitochondrial protein degradation / Transcriptional activation of mitochondrial biogenesis / single-stranded DNA binding / protein homotetramerization / mitochondrial matrix ...positive regulation of mitochondrial DNA replication / positive regulation of helicase activity / mitochondrial DNA replication / DNA unwinding involved in DNA replication / mitochondrial nucleoid / Mitochondrial protein degradation / Transcriptional activation of mitochondrial biogenesis / single-stranded DNA binding / protein homotetramerization / mitochondrial matrix / chromatin binding / protein homodimerization activity / mitochondrion / RNA binding / extracellular exosome / identical protein binding / nucleus
Similarity search - Function
Single-stranded DNA-binding protein / Single-strand binding protein family / Single-strand binding (SSB) domain profile. / Primosome PriB/single-strand DNA-binding / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / Single-stranded DNA-binding protein, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsRiccio, A.A. / Pedersen, L.C. / Bouvette, J. / Borgnia, J.M. / Copeland, W.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065078 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065078 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC ES 103326 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ.
Authors: Amanda A Riccio / Jonathan Bouvette / Lars C Pedersen / Shruti Somai / Robert C Dutcher / Mario J Borgnia / William C Copeland /
Abstract: The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. ...The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.
History
DepositionNov 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 2, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Single-stranded DNA-binding protein, mitochondrial
A: Single-stranded DNA-binding protein, mitochondrial
G: dTDNA
C: Single-stranded DNA-binding protein, mitochondrial
D: Single-stranded DNA-binding protein, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,9897
Polymers76,8745
Non-polymers1152
Water4,197233
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9620 Å2
ΔGint-35 kcal/mol
Surface area25570 Å2
MethodPISA
Unit cell
Length a, b, c (Å)135.201, 135.201, 78.081
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number95
Space group name H-MP4322
Space group name HallP4cw2c
Symmetry operation#1: x,y,z
#2: -y,x,z+3/4
#3: y,-x,z+1/4
#4: x,-y,-z+1/2
#5: -x,y,-z
#6: -x,-y,z+1/2
#7: y,x,-z+1/4
#8: -y,-x,-z+3/4

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Components

#1: Protein
Single-stranded DNA-binding protein, mitochondrial


Mass: 17708.795 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SSBP1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q04837
#2: DNA chain dTDNA


Mass: 6038.899 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Na
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 233 / 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: 2.32 Å3/Da / Density % sol: 46.93 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: 20% PEG3350 and 200 mM sodium nitrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 3, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.9→33.81 Å / Num. obs: 55728 / % possible obs: 97 % / Redundancy: 13.1 % / Biso Wilson estimate: 35.06 Å2 / CC1/2: 0.997 / Net I/σ(I): 30
Reflection shellResolution: 1.9→1.95 Å / Num. unique obs: 55728 / CC1/2: 0.813

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
HKL-3000data reduction
HKL-3000data scaling
HKL-3000phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.9→33.81 Å / SU ML: 0.2197 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 21.2076
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2072 1953 3.5 %
Rwork0.1847 53772 -
obs0.1855 55725 97.05 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 48.95 Å2
Refinement stepCycle: LAST / Resolution: 1.9→33.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3325 337 7 233 3902
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00613782
X-RAY DIFFRACTIONf_angle_d0.84715183
X-RAY DIFFRACTIONf_chiral_restr0.0584586
X-RAY DIFFRACTIONf_plane_restr0.0077603
X-RAY DIFFRACTIONf_dihedral_angle_d16.34251430
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.9-1.950.31251280.33023397X-RAY DIFFRACTION87.56
1.95-20.25341320.25053622X-RAY DIFFRACTION92.69
2-2.060.22731340.21773695X-RAY DIFFRACTION94.89
2.06-2.130.24141350.19423750X-RAY DIFFRACTION96.16
2.13-2.20.22441350.1893787X-RAY DIFFRACTION96.67
2.2-2.290.21121390.19513833X-RAY DIFFRACTION97.54
2.29-2.390.24551390.2023809X-RAY DIFFRACTION98.01
2.39-2.520.25311430.19853915X-RAY DIFFRACTION98.54
2.52-2.680.21441390.1963852X-RAY DIFFRACTION98.84
2.68-2.880.20561420.19133929X-RAY DIFFRACTION99.24
2.88-3.170.22561430.19753951X-RAY DIFFRACTION99.61
3.17-3.630.19181440.16794000X-RAY DIFFRACTION99.69
3.63-4.580.16081460.15154033X-RAY DIFFRACTION99.71
4.58-33.810.21411540.18684199X-RAY DIFFRACTION99.04
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)
16.72222202139-1.251666974181.69258967283.096444234421.592987078942.09212521512-0.278136800189-0.32860229135-0.4654146802370.2778127840430.00645373238146-0.222928930612-0.30487704326-0.4077456856180.2316183040280.298941854473-0.05538342011690.06639793287160.339078818099-0.08654511618360.39049177533645.095830253535.5970615076-20.3630621538
23.019807846531.74089253571-1.265990849728.48593888741-0.005738552146993.56963143895-0.2126147552180.239552277391-0.530661126194-0.4902181095070.202967064838-0.2563465295060.4273905624740.1069398595010.04366084432490.2703836237020.01071322741650.03105618680090.354641198275-0.1029583532840.30660570242548.801649912428.800612643-42.1558670789
38.887446272195.54512998179-0.6331321832913.72906182087-0.7503275168473.5659838202-0.09021639445170.2415334239760.0832047720406-0.1689627584460.0574925118201-0.271630384335-0.3246579852850.3933098081320.04167099307370.338103011827-0.0221244663810.06400889322220.379318976117-0.06916564273420.32182797116957.21951229639.180867977-41.7009426478
42.87897845353-0.3780973607540.7855219810724.69637863542-3.058999810967.39205972509-0.007304140200150.203358962846-0.237908821186-0.1766907238360.1108616556110.1371900023830.634000200376-0.343777319521-0.115190926750.341293860787-0.08868697984490.05195336896170.34009759215-0.1210178867940.36079685907139.710465806727.6359741718-42.6586086255
52.785450293510.212955495767-2.18095907836.5219814094-3.635285319163.52379614118-0.4799243712861.40600415618-1.16258608735-1.674381560120.271642366134-0.4478806068830.465148132343-0.6464419349780.2484165881690.54023198339-0.09413914114560.08624586203860.499794946127-0.1988673989190.36248779323647.79639290934.8548595202-51.3218066876
63.05998418992-0.08974871186270.1098402924214.55935184708-2.086190945267.664207722340.0388745837916-0.0906792698846-0.5204035691680.00753229778998-0.0274999881705-0.1656094215220.4715807504890.67454518532-0.02922656191990.326752446402-0.02074096834750.05657637463390.355323922456-0.1149958627580.37303383397753.291103666531.808401198-33.7714959066
77.64624414038-2.502418107863.210249664053.82579473592-3.812686543614.399178529810.1178167889840.247459086924-0.0500723785558-0.17952201379-0.1448663194540.1698151669440.1532118658310.0958544104512-0.07513916501460.375581898766-0.0514368616790.03791672435760.29470057488-0.1240289819590.28019373457644.064245115736.8306236379-41.1234566789
82.50182315367-0.2035839558720.6178503360531.92290331847-0.04102851679954.20222948947-0.0682662483408-0.115766819223-0.0237296928065-0.0370653235876-0.00574142450216-0.272873175153-0.2570345583880.442015263214-0.02550482225340.297613244187-0.06546996182090.05360904908770.365279892127-0.06187090492580.2917362626556.836087274541.9519727359-25.7764443429
93.8203563943-1.48409849961-3.298154791852.125235556160.4358709023563.329051172850.08906492560440.172688883560.610085131936-0.1141665372420.248979980616-1.48064476665-0.4554871217171.31355417911-0.09268035590640.417734018692-0.1205865956810.2856296504991.03226855029-0.1567815547610.87326630860376.412501241945.9449738072-24.3751526771
107.097253675842.325807772410.3747011972623.401829339661.599446718656.140502112030.0938887863346-0.664911214551-0.2162779375230.348485122598-0.0803448262196-0.2592800487510.03808792636210.5369299333830.02324175994390.2610551688930.02925587557580.01470349827640.456852268887-0.0242234967890.23313793883759.315128411739.5807309883-18.1961400612
115.513733396890.0376677134777-0.4562543531593.12750091655-1.606492971262.037959258470.01577167981410.0407879898277-0.650384662254-0.0923832587935-0.245229840812-0.5473431570360.141502589470.3560900719350.1765960883630.240407824063-0.03601219771360.02429858103450.360052961463-0.03394892388050.41078966507761.807442563837.9647560928-26.5069101661
123.17793742364-1.68129861875-3.028964120459.54927463111.954765697463.13319022348-0.8746975144030.0992474138445-0.9610640870680.792102868480.22608682087-0.7538025621921.472922863071.228319803240.4560615559050.5897826864980.1797954904170.06680988122990.8393026785420.1861995652470.60127789986460.087784040728.3930617409-14.3157592266
135.293623633241.33918513832-1.897508637181.754366803-0.1882550603622.19660917972-0.187871052598-0.692117121249-0.8178727403380.5464149682010.0504087636439-0.1298030237340.08028029634060.449164626270.1202208356840.4291548077860.02212934314470.08932678868790.379547878420.03116573187950.47017819097935.501824693933.7466293499-12.4599398561
148.31219665473-2.5581197782-1.509499824041.652682023290.05829018923280.7950165801440.4086532337880.8174187519471.0116928544-0.489611336913-0.01766195343750.2061549144-0.338527750832-0.435252878717-0.1107124635790.425116893863-0.03912897845590.06262766255680.478608495340.1006965557720.4983520065546.4449702988241.5469544092-19.5492958788
152.84814031669-0.566419412262-0.181977284971.94713511620.685527825224.88197078863-0.156440414411-0.0421670031613-0.165584188098-0.02492171653070.134295405803-0.1585914844580.4554784755410.0422500536095-0.00167075951860.246523544407-0.01749844288560.04754832950620.209841324562-0.02716166197220.32726040238310.106684954426.2740241129-3.76453295729
168.97582927459-3.797402319725.695239101434.76266011477-2.457823194743.652406920930.276090980622-0.058127474269-1.89197242081.07310025226-0.279512147042-0.4242490048380.2242516672890.723136122994-0.1889390369930.8061702630560.109758617425-0.1870717449920.4303297828850.0340230381730.85564345728921.390832675210.3706267483-4.37500044141
173.010024684592.65026344441-1.014793991093.30609209214-2.533745838313.976956425670.02149522188830.08666923764840.2457853575870.2255403598920.1077364438950.205005757511-0.346009032462-0.0343320375523-0.1582583854060.235557292754-0.02837667788240.04330336168330.301294953065-0.05075811908410.31091009177914.994818308536.6047489026-10.1843203904
183.120319978750.0599995392867-0.157664387162.7373887484-3.802675784957.62861913739-0.2263530574540.381071473343-0.457331666378-0.5651769093840.371242629694-0.3938616373020.952277511957-0.294785264481-0.1205108794270.437461484912-0.05488502075030.08529247758660.310273893466-0.1243113770880.44624682782711.363105012818.3315365597-13.7086889307
192.945904333480.1692334452652.017833658072.63512002445-1.005663411923.472243279620.07408806283580.0587308104571-0.382136563570.1422165360850.00442299665013-0.3404265937380.1476700240640.259308315973-0.07039004940240.2426943759790.004405900764780.0569447854720.281211452696-0.06663469183270.36225415932116.869734425225.2389490476-2.36536577281
201.446131587420.144011868379-0.6112410111761.55240290597-1.073082615632.49175846481-0.111546636548-0.1130110828110.1795247385120.02684371004860.11039347352-0.134101629032-0.289680211999-0.1986513722920.008756620987770.2971857134780.01077445447840.02784035101060.281720721753-0.04807442785660.3755405423558.0862924137438.19843081971.86546679816
214.882559994711.182130898080.1295394873159.19933093802-1.425083131574.31047968883-0.0862766376379-0.234265573645-0.2757225700650.3005417796940.0670882051806-0.3742176631090.1605747257460.08081621309310.04144185091580.2245338084650.0386559909995-0.006761643983360.366967669777-0.01583171853880.29966589051913.783491352731.07308439712.4448377026
225.413651127650.2005137667370.06037713388343.23991732494-0.5962154063463.00295116366-0.0494636364177-0.1221376486950.2998973076860.1162663157120.0302479034987-0.467459646319-0.2530832507330.37319810615-0.01713994552680.232892776333-0.00543140097052-0.03437377958150.266068688294-0.04957540672390.36897300602513.62324311239.39016108449.00270751707
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'B' and (resid 26 through 32 )BA26 - 321 - 7
22chain 'B' and (resid 33 through 53 )BA33 - 538 - 28
33chain 'B' and (resid 54 through 89 )BA54 - 8929 - 54
44chain 'B' and (resid 90 through 101 )BA90 - 10155 - 66
55chain 'B' and (resid 102 through 106 )BA102 - 10667 - 71
66chain 'B' and (resid 107 through 130 )BA107 - 13072 - 88
77chain 'B' and (resid 131 through 142 )BA131 - 14289 - 100
88chain 'A' and (resid 26 through 46 )AC26 - 461 - 21
99chain 'A' and (resid 47 through 53 )AC47 - 5322 - 28
1010chain 'A' and (resid 54 through 106 )AC54 - 10629 - 66
1111chain 'A' and (resid 107 through 142 )AC107 - 14267 - 102
1212chain 'G' and (resid 3 through 7 )GD3 - 7
1313chain 'G' and (resid 8 through 12 )GD8 - 12
1414chain 'G' and (resid 13 through 19 )GD13 - 19
1515chain 'C' and (resid 26 through 46 )CE26 - 461 - 21
1616chain 'C' and (resid 47 through 53 )CE47 - 5322 - 28
1717chain 'C' and (resid 54 through 89 )CE54 - 8929 - 55
1818chain 'C' and (resid 90 through 106 )CE90 - 10656 - 72
1919chain 'C' and (resid 107 through 141 )CE107 - 14173 - 107
2020chain 'D' and (resid 22 through 43 )DF22 - 431 - 22
2121chain 'D' and (resid 44 through 89 )DF44 - 8923 - 53
2222chain 'D' and (resid 90 through 143 )DF90 - 14354 - 107

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  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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