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- PDB-6g3b: AvaII restriction endonuclease in complex with an RNA/DNA hybrid -

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Basic information

Entry
Database: PDB / ID: 6g3b
TitleAvaII restriction endonuclease in complex with an RNA/DNA hybrid
Components
  • DNA (5'-D(*CP*CP*AP*TP*GP*GP*TP*CP*CP*TP*A)-3')
  • RNA (5'-R(P*GP*UP*AP*GP*GP*AP*CP*CP*AP*UP*G)-3')
  • Type II site-specific deoxyribonuclease
KeywordsHYDROLASE / RESTRICTION ENDONUCLEASE / SCANNING COMPLEX / RNA/DNA HYBRID / RNA/DNA HETERODUPLEX / A-DNA
Function / homology
Function and homology information


type II site-specific deoxyribonuclease activity / DNA restriction-modification system / DNA binding
Similarity search - Function
Type II restriction endonuclease SinI / SinI restriction endonuclease
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Type II site-specific deoxyribonuclease
Similarity search - Component
Biological speciesNostoc sp. PCC 7120 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.8 Å
AuthorsKisiala, M. / Kowalska, M. / Czapinska, H. / Bochtler, M.
Funding support Poland, 6items
OrganizationGrant numberCountry
UMO-2011/02/A/NZ1/00052 Poland
UMO-2014/13/B/NZ1/03991 Poland
UMO-2014/14/M/NZ5/00558 Poland
0295/B/PO1/2008/34 Poland
N N301 425038 Poland
N N301 028 934 Poland
Citation
Journal: Nucleic Acids Res. / Year: 2020
Title: Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation
Authors: Kisiala, M. / Kowalska, M. / Pastor, M. / Korza, H. / Czapinska, H. / Bochtler, M.
#1: Journal: Nucleic Acids Res. / Year: 2010
Title: Sequence-specific cleavage of RNA by Type II restriction enzymes.
Authors: Murray, I.A. / Stickel, S.K. / Roberts, R.J.
#2: Journal: J. Biol. Chem. / Year: 2005
Title: Crystal structures of type II restriction endonuclease EcoO109I and its complex with cognate DNA.
Authors: Hashimoto, H. / Shimizu, T. / Imasaki, T. / Kato, M. / Shichijo, N. / Kita, K. / Sato, M.
History
DepositionMar 24, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2019Provider: repository / Type: Initial release
Revision 1.1May 20, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type II site-specific deoxyribonuclease
B: Type II site-specific deoxyribonuclease
C: RNA (5'-R(P*GP*UP*AP*GP*GP*AP*CP*CP*AP*UP*G)-3')
D: DNA (5'-D(*CP*CP*AP*TP*GP*GP*TP*CP*CP*TP*A)-3')


Theoretical massNumber of molelcules
Total (without water)61,1794
Polymers61,1794
Non-polymers00
Water4,990277
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7550 Å2
ΔGint-61 kcal/mol
Surface area25350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)37.680, 104.050, 78.290
Angle α, β, γ (deg.)90.00, 93.27, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Type II site-specific deoxyribonuclease / / AvaII restriction endonuclease


Mass: 27161.797 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. PCC 7120 (bacteria) / Gene: alr0933 / Production host: Escherichia coli (E. coli) / Variant (production host): ER2566 / References: UniProt: Q8YYB7
#2: RNA chain RNA (5'-R(P*GP*UP*AP*GP*GP*AP*CP*CP*AP*UP*G)-3')


Mass: 3546.178 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*CP*CP*AP*TP*GP*GP*TP*CP*CP*TP*A)-3')


Mass: 3309.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 277 / 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: 2.65 Å3/Da / Density % sol: 53.59 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 10% W/V PEG 20000, 20% V/V PEG MME 550, 0.02 M Na-Formate; 0.02 M NH4-Acetate; 0.02 M Na3-Citrate; 0.02 M NaK-Tartrate (racemic); 0.02 M Na-Oxamate AND 0.1 M MOPS/HEPES-NA, PH 7.5, GLYCEROL ...Details: 10% W/V PEG 20000, 20% V/V PEG MME 550, 0.02 M Na-Formate; 0.02 M NH4-Acetate; 0.02 M Na3-Citrate; 0.02 M NaK-Tartrate (racemic); 0.02 M Na-Oxamate AND 0.1 M MOPS/HEPES-NA, PH 7.5, GLYCEROL WAS ADDED FOR CRYOPROTECTION
PH range: 7.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 1.2782 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 29, 2013
RadiationMonochromator: KMC-1 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.2782 Å / Relative weight: 1
ReflectionResolution: 1.8→52.025 Å / Num. obs: 53209 / % possible obs: 95.4 % / Redundancy: 2.6 % / Biso Wilson estimate: 42.229 Å2 / CC1/2: 0.998 / Rrim(I) all: 0.051 / Rsym value: 0.041 / Net I/σ(I): 12.96
Reflection shellResolution: 1.8→1.85 Å / Redundancy: 2.6 % / Mean I/σ(I) obs: 1.86 / Num. unique obs: 4000 / CC1/2: 0.57 / Rrim(I) all: 0.798 / Rsym value: 0.638 / % possible all: 97.2

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
XDSdata reduction
XSCALEdata scaling
SHELXDEphasing
ARP/wARPmodel building
RefinementMethod to determine structure: SAD / Resolution: 1.8→52.025 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 21.85
Details: The register and the strand assignment of the RNA/DNA heteroduplex is unsure. It is plausible that the observed density corresponds to the average of the heteroduplexes bound in multiple ...Details: The register and the strand assignment of the RNA/DNA heteroduplex is unsure. It is plausible that the observed density corresponds to the average of the heteroduplexes bound in multiple different registers. The sequence assignment is based on the known sequence specificity of AvaII endonuclease but remains very tentative. The density for the 166-176 fragments of both enzyme protomers is very poor. TLS refinement has been used. Hydrogens have been added in the riding positions. Group B-factor refinement has been used for the RNA/DNA molecule.
RfactorNum. reflection% reflectionSelection details
Rfree0.2021 2665 5.01 %RANDOM
Rwork0.1675 ---
obs0.1694 53174 95.38 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.8→52.025 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3587 454 0 277 4318
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0244880
X-RAY DIFFRACTIONf_angle_d1.3076742
X-RAY DIFFRACTIONf_dihedral_angle_d14.911917
X-RAY DIFFRACTIONf_chiral_restr0.056752
X-RAY DIFFRACTIONf_plane_restr0.008715
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.83270.31041260.28652700X-RAY DIFFRACTION97
1.8327-1.8680.26961410.26372700X-RAY DIFFRACTION97
1.868-1.90610.30281320.24492709X-RAY DIFFRACTION97
1.9061-1.94750.28321600.22692674X-RAY DIFFRACTION97
1.9475-1.99280.2121530.20292639X-RAY DIFFRACTION95
1.9928-2.04270.26071240.19492753X-RAY DIFFRACTION99
2.0427-2.09790.21091500.18922743X-RAY DIFFRACTION98
2.0979-2.15960.20521320.18422697X-RAY DIFFRACTION98
2.1596-2.22940.21671420.17952732X-RAY DIFFRACTION97
2.2294-2.3090.24251380.1712659X-RAY DIFFRACTION96
2.309-2.40150.21871390.1682678X-RAY DIFFRACTION96
2.4015-2.51080.22311480.16912709X-RAY DIFFRACTION97
2.5108-2.64310.21841300.16892651X-RAY DIFFRACTION96
2.6431-2.80870.21141420.16812629X-RAY DIFFRACTION95
2.8087-3.02560.23861290.17512578X-RAY DIFFRACTION92
3.0256-3.330.22971430.1632627X-RAY DIFFRACTION95
3.33-3.81170.18061410.14452605X-RAY DIFFRACTION93
3.8117-4.80190.1661520.13682503X-RAY DIFFRACTION90
4.8019-52.04670.17691430.17172523X-RAY DIFFRACTION89
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)
10.843-0.10110.08680.61690.64461.04630.0222-0.27070.0134-0.02790.0039-0.0322-0.01940.0524-00.2371-0.02670.01750.23170.00940.212423.927929.743454.7461
20.48140.2934-0.38150.8685-0.65161.23990.0368-0.24290.02640.0062-0.09130.0074-0.03630.10960.00010.17640.0174-0.01780.2279-0.00030.204323.6098-6.400353.4922
33.64693.06691.91013.07620.86482.8627-0.339-0.773-0.5590.42350.4745-0.31090.00330.0277-0.02040.7387-0.1018-0.00961.3491-0.04171.327223.725812.139458.1172
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A'
2X-RAY DIFFRACTION2chain 'B'
3X-RAY DIFFRACTION3chain 'C' or chain 'D'

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