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- PDB-5n35: Gadolinium phased PBP2 (SSO6202) at 2.2 Ang -

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Basic information

Entry
Database: PDB / ID: 5n35
TitleGadolinium phased PBP2 (SSO6202) at 2.2 Ang
ComponentsPolB1 Binding Protein 2 (PBP2)
KeywordsPOLYMERASE BINDING PROTEIN / PolB1 binding protein 2 / Archaeal DNA Polymerase Holoenzyme / Protein binding
Function / homologyGADOLINIUM ATOM / NITRATE ION / Uncharacterized protein
Function and homology information
Biological speciesSulfolobus solfataricus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.24 Å
AuthorsYan, J. / Beattie, T.R. / Rojas, A.L. / Schermerhorn, K. / Gristwood, T. / Trinidad, J.C. / Albers, S.V. / Roversi, P. / Gardner, A.F. / Abrescia, N.G.A. / Bell, S.D.
Funding support Spain, 1items
OrganizationGrant numberCountry
MINECOBFU2015-64541-R Spain
CitationJournal: Nat Commun / Year: 2017
Title: Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.
Authors: Jiangyu Yan / Thomas R Beattie / Adriana L Rojas / Kelly Schermerhorn / Tamzin Gristwood / Jonathan C Trinidad / Sonja V Albers / Pietro Roversi / Andrew F Gardner / Nicola G A Abrescia / Stephen D Bell /
Abstract: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family ...Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
History
DepositionFeb 8, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Structure summary / Category: audit_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: PolB1 Binding Protein 2 (PBP2)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)9,3417
Polymers8,6881
Non-polymers6536
Water54030
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1070 Å2
ΔGint-15 kcal/mol
Surface area3940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.150, 62.150, 35.680
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein PolB1 Binding Protein 2 (PBP2)


Mass: 8688.045 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus solfataricus (archaea) / Gene: SSOP1_0579, SULA_1676, SULB_1677, SULC_1675 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: A0A0E3GTJ4
#2: Chemical ChemComp-GD / GADOLINIUM ATOM


Mass: 157.250 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Gd
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-NO3 / NITRATE ION / Nitrate


Mass: 62.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NO3
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 30 / 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: 2.29 Å3/Da / Density % sol: 46.28 %
Preparation: FOR ANALYSIS, PLEASE, USE THE NATIVE HIGH-RESOLUTION 1.35 Ang STRUCTURE: PDB 5N41
Crystal growTemperature: 294.15 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Protein: 42.7 mg/ml in 20 mM Hepes pH 7.5, 0.3 M NaCl, 1mM MgCl2 and 1mM b-ME. Crystallization buffer: 0.2 M NaNO3, 20% PEG 3350

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21001
31001
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONESRF ID2911.71076
SYNCHROTRONESRF ID2921.71145
SYNCHROTRONESRF ID2931.70371
Detector
TypeIDDetectorDate
DECTRIS PILATUS 6M1PIXELNov 22, 2012
DECTRIS PILATUS 6M2PIXELNov 22, 2012
DECTRIS PILATUS 6M3PIXELNov 22, 2016
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1MADMx-ray1
2MADMx-ray2
3MADMx-ray3
Radiation wavelength
IDWavelength (Å)Relative weight
11.710761
21.711451
31.703711
ReflectionResolution: 2.2→31.1 Å / Num. obs: 3963 / % possible obs: 99 % / Redundancy: 17 % / Biso Wilson estimate: 30.9 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 31.6
Reflection shellHighest resolution: 2.2 Å / Redundancy: 8.7 % / Rmerge(I) obs: 0.07 / Mean I/σ(I) obs: 1.3 / % possible all: 90.7

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Processing

Software
NameVersionClassification
BUSTER2.11.2refinement
XDSdata reduction
SCALAdata scaling
SHARPphasing
RefinementMethod to determine structure: MAD / Resolution: 2.24→31.08 Å / Cor.coef. Fo:Fc: 0.9153 / Cor.coef. Fo:Fc free: 0.8809 / SU R Cruickshank DPI: 0.304 / Cross valid method: FREE R-VALUE / σ(F): 0 / SU R Blow DPI: 0.317 / SU Rfree Blow DPI: 0.24 / SU Rfree Cruickshank DPI: 0.235
Details: The refinement was carried out with the data from remote 1.
RfactorNum. reflection% reflectionSelection details
Rfree0.2748 171 4.57 %RANDOM
Rwork0.2271 ---
obs0.2294 3740 93.62 %-
Displacement parametersBiso mean: 37.15 Å2
Baniso -1Baniso -2Baniso -3
1--1.6342 Å20 Å20 Å2
2---1.6342 Å20 Å2
3---3.2683 Å2
Refine analyzeLuzzati coordinate error obs: 0.401 Å
Refinement stepCycle: LAST / Resolution: 2.24→31.08 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms468 0 24 30 522
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.01521HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.29710HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d195SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes15HARMONIC2
X-RAY DIFFRACTIONt_gen_planes68HARMONIC5
X-RAY DIFFRACTIONt_it521HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion2.42
X-RAY DIFFRACTIONt_other_torsion19.55
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion67SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact674SEMIHARMONIC4
LS refinement shellResolution: 2.24→2.5 Å / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.3961 42 4.81 %
Rwork0.3766 832 -
all0.3776 874 -
obs--93.62 %

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