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- PDB-5dwa: Crystal structure of pre-specific restriction endonuclease AgeI-D... -

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Basic information

Entry
Database: PDB / ID: 5dwa
TitleCrystal structure of pre-specific restriction endonuclease AgeI-DNA complex
Components
  • DNA (5'-D(*TP*CP*GP*AP*CP*CP*GP*GP*TP*CP*G*)-3')
  • Type-2 restriction enzyme AgeI
KeywordsHYDROLASE / restriction endonuclease / PD-(D/E)XK nuclease / protein-DNA complex
Function / homologyBsaWI restriction endonuclease type 2 / BsaWI restriction endonuclease type 2 / type II site-specific deoxyribonuclease / type II site-specific deoxyribonuclease activity / DNA restriction-modification system / DNA / DNA (> 10) / Type II restriction enzyme AgeI
Function and homology information
Biological speciesThalassobius gelatinovorus (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / molecular replacement / Resolution: 1.5 Å
AuthorsTamulaitiene, G. / Jovaisaite, V. / Grazulis, S. / Siksnys, V.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaMIP-41/2013Lithuania
CitationJournal: Nucleic Acids Res. / Year: 2017
Title: Restriction endonuclease AgeI is a monomer which dimerizes to cleave DNA.
Authors: Tamulaitiene, G. / Jovaisaite, V. / Tamulaitis, G. / Songailiene, I. / Manakova, E. / Zaremba, M. / Grazulis, S. / Xu, S.Y. / Siksnys, V.
History
DepositionSep 22, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Oct 5, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2017Group: Database references
Revision 1.2May 3, 2017Group: Database references
Revision 1.3Jul 17, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.4May 8, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / diffrn_radiation_wavelength
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type-2 restriction enzyme AgeI
B: Type-2 restriction enzyme AgeI
C: DNA (5'-D(*TP*CP*GP*AP*CP*CP*GP*GP*TP*CP*G*)-3')
D: DNA (5'-D(*TP*CP*GP*AP*CP*CP*GP*GP*TP*CP*G*)-3')


Theoretical massNumber of molelcules
Total (without water)67,8554
Polymers67,8554
Non-polymers00
Water9,440524
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7710 Å2
ΔGint-42 kcal/mol
Surface area22680 Å2
MethodPISA
Unit cell
Length a, b, c (Å)66.003, 79.806, 101.655
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Type-2 restriction enzyme AgeI / R.AgeI / Endonuclease AgeI / Type II restriction enzyme AgeI


Mass: 30577.520 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thalassobius gelatinovorus (bacteria) / Gene: ageIR / Plasmid: pRRS-AgeIRM / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566
References: UniProt: Q9KHV6, type II site-specific deoxyribonuclease
#2: DNA chain DNA (5'-D(*TP*CP*GP*AP*CP*CP*GP*GP*TP*CP*G*)-3')


Mass: 3350.185 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 524 / 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

Crystal
IDDensity Matthews (Å3/Da)Density % sol (%)
11.9737.65
21.9737.65
Crystal grow
Temperature (K)Crystal-IDMethodpHDetails
2911vapor diffusion, sitting drop5.50.2 M NaCl, 0.1 M Bis-Tris (pH 5.5), 19% PEG3350
2912vapor diffusion, sitting drop5.50.2 M NaCl, 0.1 M Bis-Tris (pH 5.5), 19% PEG3350

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21002
Diffraction source
SourceSiteBeamlineTypeIDWavelength (Å)
SYNCHROTRONEMBL/DESY, HAMBURG X1210.99985
ROTATING ANODERIGAKU MICROMAX-007 HF21.5418
Detector
TypeIDDetectorDateDetails
MARMOSAIC 225 mm CCD1CCDDec 8, 2008mirrors
RIGAKU RAXIS IV++2IMAGE PLATEFeb 9, 2009
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
2SINGLE WAVELENGTHMx-ray2
Radiation wavelength
IDWavelength (Å)Relative weight
10.999851
21.54181
ReflectionRedundancy: 14.3 % / Number: 245909 / Rmerge(I) obs: 0.071 / Rsym value: 0.071 / D res high: 2.6 Å / D res low: 43.153 Å / Num. obs: 17243 / % possible obs: 100
Diffraction reflection shell
Highest resolution (Å)Lowest resolution (Å)IDRmerge(I) obsRsym valueRedundancy
8.2243.1510.0390.03912.4
5.818.2210.0430.04313.9
4.755.8110.0490.04914.2
4.114.7510.0480.04814.4
3.684.1110.0570.05714.4
3.363.6810.0690.06914.4
3.113.3610.0940.09414.5
2.913.1110.1270.12714.5
2.742.9110.1770.17714.4
2.62.7410.2350.23514.1
ReflectionResolution: 1.5→79.809 Å / Num. all: 84560 / Num. obs: 84560 / % possible obs: 97.8 % / Redundancy: 8.2 % / Biso Wilson estimate: 15.32 Å2 / Rmerge(I) obs: 0.062 / Rpim(I) all: 0.017 / Rrim(I) all: 0.066 / Rsym value: 0.062 / Net I/av σ(I): 6.903 / Net I/σ(I): 24.7 / Num. measured all: 694292
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRsym valueNet I/σ(I) obs% possible all
1.5-1.5850.1973.861889124580.1070.1974.7100
1.58-1.6850.1484.859251118620.0780.1487.3100
1.68-1.794.80.1116.253679111320.0590.11110.699.9
1.79-1.944.40.0897.34428699770.0490.08914.496.3
1.94-2.124.10.0827.53556086820.0470.08219.690.9
2.12-2.376.30.1065.95084980100.040.10625.892.5
2.37-2.7417.40.0867.613472677270.0210.08639.5100
2.74-3.3517.20.069.711322765710.0150.0656100
3.35-4.7417.50.05510.49026451600.0130.05576.8100
4.74-79.809170.05210.45056129810.0130.05279.899.9

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Phasing

Phasing
Method
SAD
molecular replacement
Phasing MAD setHighest resolution: 3 Å / Lowest resolution: 20 Å
Phasing MAD set shell
IDResolution (Å)
111.71-20
18.28-11.71
16.4-8.28
15.22-6.4
14.4-5.22
13.81-4.4
13.36-3.81
13-3.36
Phasing MAD set site

Atom type symbol: HG

IDB isoFract xFract yFract zOccupancyOccupancy iso
1-6.4790.5141.4480.1660.3380.481
20.9710.5620.9940.8620.350.479
Phasing MAD shell
Resolution (Å)FOM FOM acentricFOM centricReflectionReflection acentricReflection centric
11.71-200.5680.5110.63722112299
8.28-11.710.5360.530.551375267108
6.4-8.280.5420.5320.575646493153
5.22-6.40.5050.4920.5621008819189
4.4-5.220.4130.4080.44314131184229
3.81-4.40.380.3690.44919241654270
3.36-3.810.3460.340.38625002192308
3-3.360.3460.3350.43731372789348
Phasing MR
Highest resolutionLowest resolution
Rotation1.74 Å55.36 Å
Translation1.74 Å55.36 Å
Phasing dmMethod: Solvent flattening and Histogram matching / Reflection: 11228
Phasing dm shell
Resolution (Å)Delta phi finalFOM Reflection
8.77-10063.80.583507
6.89-8.7752.10.79503
5.99-6.8953.70.743508
5.41-5.9962.30.757510
5-5.4159.10.756507
4.7-554.50.746506
4.45-4.7520.76514
4.25-4.4560.20.69506
4.09-4.2570.60.647505
3.94-4.0970.70.62506
3.81-3.9465.50.637505
3.7-3.8167.90.625505
3.6-3.7690.583501
3.51-3.6700.636501
3.43-3.5174.60.613512
3.35-3.4370.90.61505
3.29-3.3570.90.543507
3.22-3.2971.70.583532
3.16-3.2270.50.631523
3.1-3.1675.80.601548
2.99-3.175.10.451017

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Processing

Software
NameVersionClassification
MOSFLM6.2.6data reduction
SCALA3.2.19data scaling
MLPHARE6phasing
MOLREP6phasing
DM6phasing
Coot0.6.1model building
PHENIX1.8.3refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: SAD / Resolution: 1.5→40.27 Å / SU ML: 0.14 / Cross valid method: FREE R-VALUE / σ(F): 1.28 / Phase error: 17.6 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.1852 8036 10.06 %Random selection
Rwork0.1517 143711 --
obs0.155 84472 96.19 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 87.47 Å2 / Biso mean: 22.6389 Å2 / Biso min: 8.46 Å2
Refinement stepCycle: final / Resolution: 1.5→40.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4271 444 0 524 5239
Biso mean---31.35 -
Num. residues----578
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0065110
X-RAY DIFFRACTIONf_angle_d1.0227043
X-RAY DIFFRACTIONf_chiral_restr0.04763
X-RAY DIFFRACTIONf_plane_restr0.005868
X-RAY DIFFRACTIONf_dihedral_angle_d16.7261948
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.5-1.5170.25355350.19444949548499
1.517-1.53490.22455820.179449305512100
1.5349-1.55360.21755610.16774919548099
1.5536-1.57330.21045420.16344976551899
1.5733-1.5940.20046020.16244957555999
1.594-1.61580.19325280.15414946547499
1.6158-1.63890.20516140.14934823543799
1.6389-1.66340.21795670.15444922548999
1.6634-1.68940.21445930.14444934552799
1.6894-1.71710.21095060.14474878538499
1.7171-1.74670.19245360.14925048558499
1.7467-1.77840.18955410.14114905544698
1.7784-1.81260.20275480.1384862541098
1.8126-1.84960.19475470.14184764531196
1.8496-1.88990.18355780.13714673525194
1.8899-1.93380.18334570.13434600505792
1.9338-1.98220.21275100.14864421493189
1.9822-2.03580.18154560.15434366482287
2.0358-2.09570.19994560.16024245470185
2.0957-2.16330.18784600.15744215467584
2.1633-2.24060.19434650.14984135460083
2.2406-2.33030.18214880.15594340482887
2.3303-2.43640.20135060.155650485554100
2.4364-2.56480.19065810.152449435524100
2.5648-2.72550.18435870.160749455532100
2.7255-2.93580.17755310.162350065537100
2.9358-3.23120.18115810.166749825563100
3.2312-3.69840.1875780.149449685546100
3.6984-4.65850.15785030.129350285531100
4.6585-40.2850.15215330.155949835516100

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