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- PDB-8ik4: Structure of DNA binding domain of McrBC endonuclease bound to he... -

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Basic information

Entry
Database: PDB / ID: 8ik4
TitleStructure of DNA binding domain of McrBC endonuclease bound to hemimethylated DNA: L68F mutant
Components
  • DNA (5'-D(*AP*GP*AP*(5CM)P*CP*GP*GP*TP*AP*G)-3')
  • DNA (5'-D(*CP*TP*AP*CP*CP*GP*GP*TP*CP*T)-3')
  • Type IV methyl-directed restriction enzyme EcoKMcrB subunit
KeywordsDNA BINDING PROTEIN / McrB-NTD / 5-methylcytosine / restriction endonuclease
Function / homology
Function and homology information


restriction endodeoxyribonuclease activity / endonuclease complex / double-stranded methylated DNA binding / hemi-methylated DNA-binding / DNA catabolic process / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / DNA restriction-modification system / endonuclease activity / GTPase activity / GTP binding ...restriction endodeoxyribonuclease activity / endonuclease complex / double-stranded methylated DNA binding / hemi-methylated DNA-binding / DNA catabolic process / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / DNA restriction-modification system / endonuclease activity / GTPase activity / GTP binding / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding
Similarity search - Function
Type IV methyl-directed restriction enzyme EcoKMcrB subunit, DNA-binding domain / MrcB-like, N-terminal domain / : / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Type IV methyl-directed restriction enzyme EcoKMcrB subunit
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
DNA molecule (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsAdhav, V.A. / Saikrishnan, K.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: To Be Published
Title: Structural basis of target recognition by the DNA binding domain of McrBC
Authors: Adhav, V.A. / Saikrishnan, K.
History
DepositionFeb 28, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type IV methyl-directed restriction enzyme EcoKMcrB subunit
B: Type IV methyl-directed restriction enzyme EcoKMcrB subunit
C: DNA (5'-D(*AP*GP*AP*(5CM)P*CP*GP*GP*TP*AP*G)-3')
D: DNA (5'-D(*CP*TP*AP*CP*CP*GP*GP*TP*CP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,7656
Polymers47,3464
Non-polymers4182
Water4,017223
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)36.099, 70.279, 144.179
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Type IV methyl-directed restriction enzyme EcoKMcrB subunit / EcoKMcrBC / 5-methylcytosine-specific restriction enzyme B


Mass: 19693.938 Da / Num. of mol.: 2 / Mutation: L68F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: mcrB, rglB, b4346, JW5871 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P15005, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: DNA chain DNA (5'-D(*AP*GP*AP*(5CM)P*CP*GP*GP*TP*AP*G)-3')


Mass: 4030.650 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#3: DNA chain DNA (5'-D(*CP*TP*AP*CP*CP*GP*GP*TP*CP*T)-3')


Mass: 3927.561 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#4: Chemical ChemComp-BTB / 2-[BIS-(2-HYDROXY-ETHYL)-AMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / BIS-TRIS BUFFER


Mass: 209.240 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H19NO5 / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 223 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 1.93 Å3/Da / Density % sol: 36.32 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 0.1-0.2 M Bis-Tris pH 5.5 and 12-24% PEG 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Oct 20, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.1→70.28 Å / Num. obs: 21681 / % possible obs: 97.9 % / Redundancy: 4.4 % / Biso Wilson estimate: 25.96 Å2 / CC1/2: 0.993 / Rmerge(I) obs: 0.117 / Net I/σ(I): 7.7
Reflection shellResolution: 2.1→2.16 Å / Redundancy: 4.2 % / Rmerge(I) obs: 0.678 / Mean I/σ(I) obs: 2 / Num. unique obs: 1702 / CC1/2: 0.527 / % possible all: 96.9

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
iMOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3SSE

3sse


Resolution: 2.1→50.32 Å / SU ML: 0.2204 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.3372
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2464 1125 5.2 %
Rwork0.1963 20509 -
obs0.1989 21634 97.28 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 33.28 Å2
Refinement stepCycle: LAST / Resolution: 2.1→50.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2507 405 28 223 3163
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00213549
X-RAY DIFFRACTIONf_angle_d0.48244985
X-RAY DIFFRACTIONf_chiral_restr0.0363516
X-RAY DIFFRACTIONf_plane_restr0.002502
X-RAY DIFFRACTIONf_dihedral_angle_d21.1546742
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.20.3241170.28612531X-RAY DIFFRACTION97.1
2.2-2.310.28431440.26052425X-RAY DIFFRACTION95.11
2.31-2.460.30011470.24312560X-RAY DIFFRACTION98.36
2.46-2.650.31641330.23262562X-RAY DIFFRACTION98.47
2.65-2.910.2651390.22372558X-RAY DIFFRACTION97.89
2.91-3.330.25291550.19862528X-RAY DIFFRACTION96.37
3.33-4.20.20591520.14982608X-RAY DIFFRACTION98.01
4.2-50.320.19911380.16132737X-RAY DIFFRACTION96.9
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)
10.481278717753-1.23871109578-0.1831214686443.586714647641.865010434825.0549050042-0.227458990009-0.696458748868-0.4037337109611.049624877960.01787556772170.5105007205650.3199757386850.1837683046210.0133064239790.389910984207-0.1045354249550.1327646099440.6040045522760.1261418552040.280076146204-6.23902618392-4.0247405393228.987931513
27.21328934615-2.87938617384-2.169840495244.399997860021.186459621066.27445670884-0.0810604368433-0.2719542242260.6917068094380.310119147488-0.119308950343-0.140181531803-0.00512304027562-0.5110687566940.191008337640.1597002036-0.0598823223697-0.02894046177280.318550782826-0.01674561934780.305754452466-6.854127674656.7325013506717.3622357105
35.779867136772.31372683102-0.8905628183467.25245646325-3.34450281563.584947025730.0267150399533-0.7505892007670.1601631504220.5855778781810.289837222904-0.069367719997-0.306966635275-0.227819329483-0.2540444272780.252531323806-0.0114399369336-0.000579088395570.502864174674-0.05650165569690.1964348953873.2262149320210.334000996425.2218059211
41.74740600924-0.2251509183890.4061748591187.163815484630.5530405399632.12815016711-0.0185721025025-1.1534717118-0.4570222602151.000689822210.2709606807070.1819897824260.281536880354-0.596722166455-0.100639804020.2924696831740.00644608462007-0.02611474000170.8760157709140.2922960710490.3831582401528.12845179992-2.386747466728.6319113116
50.516877441160.184062221191-0.01861417907831.172049094271.034007431373.37044557649-0.256246401479-0.269705134735-0.3516782577240.2548539194760.192160266628-0.1818870501690.2877459184760.09121023711870.1288189775250.2339556892730.1081753003320.02272222145590.304441686574-0.05863658422770.282399188194.04739195453-0.518574264104-3.67605706778
60.498114857826-0.3827294304150.6163799902370.47368088036-0.4321900767552.746141736070.163273081528-0.0281904716576-0.221738773823-0.311929321973-0.05450343928910.1233420614560.438760407159-0.125431903639-0.2661134475650.41330296977-0.15162078575-0.2078835143560.4931610677440.1930175404210.09900802931194.05794980103-0.361388236921-3.69814306146
76.02031171931.899776902270.6829366245864.33238668106-0.3056021920074.49036147869-0.2336655915980.1672517249940.0672794748513-0.06219863639540.3424604516640.469352732053-0.208726497582-0.074706432471-0.08084638883570.1704180852880.0378535258175-0.03522523529810.2133233655020.03363712640870.245040431627-8.118309054294.13870014107-27.3266559977
83.23464307947-1.12266457250.525281652762.546421751830.6248105514475.14355986272-0.173849850118-0.1735244068180.2885962103420.000246660616207-0.03100561403220.294180823858-0.445459925382-0.670722087430.01375800440330.1563907348640.03403108882290.004269351755230.22393373597-0.0140836485030.186653625335-9.315560924943.56565991084-17.4151293266
93.05489576657-0.2852754593040.4202078849561.32161303422-0.3140489490323.10296111403-0.0933671384928-0.169548628415-0.124318934835-0.07225037598390.131492961070.1607458331430.115175597396-0.215636789679-0.0009348254252480.175652570421-0.01198668571170.003704995625880.1271351304710.01938100570870.181546678262-3.15818789544-3.20503514619-20.750677476
102.759465545040.350402468440.03265068074692.33232551604-1.100330155372.4127542146-0.1088013993980.09678560846450.02806604532620.05041032616620.09796691710890.009673463622870.1573351611170.07822581232550.003230129676110.2027268130940.01405262399780.003602552431910.119553304854-0.03316453999830.1152002970954.14107271835-3.67783859811-24.308246416
111.85464188619-0.839087826498-0.6003991303443.7946204005-1.556511197633.320791603230.0715113330237-0.324708281863-0.5824352394280.3736122926990.362825763749-1.1826498570.7799937138230.616849053962-0.2793389786980.3602645967860.0836773002283-0.01244482220740.1195286935790.01589376530310.4275899884659.56006336453-13.682684732-19.751590504
125.044464893896.086061552181.194996062367.802087195272.094737663851.83095738308-0.09800582220121.26491198289-1.10634981347-1.487472718380.265744220504-0.7546218627290.7115097138950.118353173428-0.06685776222740.5946903817060.06033887023010.01180336672850.469688814895-0.138841472290.3753488985513.9088519423-11.2246992196-34.1227298853
133.380162778280.1490820908671.00541583461.25538085884-0.08698125284273.03157552628-0.01496451322460.2867131027230.0611495274337-0.109691423645-0.0624614789720.01390362665560.01690092322740.4921001052610.09100162427770.1912565615020.0250025019303-0.01895610804590.2221165970930.01472291377760.21281086841711.01397150093.08658560768-28.7570571197
143.2104542975-0.668656925088-0.6302962845213.269731429831.143344032232.263303027720.08881337730290.0935883495975-0.28117144256-0.44899331402-0.0501778384655-0.215408428860.5270033148760.13349806687-0.02456198497720.281995204513-0.0533534619779-0.04745277234710.1699451488870.01359384011980.2332311046080.756169755256-12.1271177911-32.3456298071
155.43778738073-1.948852676092.274668768243.0241711651-0.865754899034.72797185566-0.113919179863-0.473320528240.7330445811360.136323744931-0.22217073638-0.439009036563-0.1657623711780.4342278188780.3156472981590.161591227454-0.0670154619910.007810760708090.368475203059-0.009073778663610.24145561687716.40112039998.4335649765719.0939078906
163.458181416980.01848192295160.2229131452243.6224701778-2.589425101673.978707344060.0155268639735-0.00816815085683-0.2425967642790.0667163963978-0.225530399862-0.408952870453-0.1424742421630.6109965903070.2186707763390.178964522431-0.0341541508426-0.005475954138530.22569516156-0.007611298114190.19161455873816.1939170730.4392287049614.2983226652
172.67975082075-0.532298457208-0.06486789390741.33957502041-0.07171233988483.300122097930.1066903726610.0528019336376-0.511023741213-0.0371847697879-0.0443802924724-0.0154302952110.0959254238449-0.0330661125038-0.04894221394560.146522598634-0.0412183039813-0.03065408999880.148700974674-0.004771336118190.2121438626196.78588359959-0.069290286815313.022413422
183.25511443492-0.819914863161.452564403014.18181378937-0.4742032847835.28197004776-0.16360945223-0.2499154931050.00844283100527-0.2291610162930.1567754079750.152408736017-0.435093259503-0.329851513327-0.07614328353640.236799327762-0.006304167775570.005290164705680.2036583182030.006076687922480.2015478931191.356187811767.2352929298418.9042327371
190.8227219981450.8345398464141.026730090850.8875643909660.6242793987694.749921405070.2180580683550.0783772689123-0.792352932001-0.3317657561250.3878581525960.4581731206410.772095919183-0.643030623054-0.1141356185490.372379585122-0.142549744051-0.1544612777110.4156973870040.01240845582470.545118691917-1.73761951475-9.8960666558114.6789304072
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'B' and (resid 93 through 101 )BB93 - 10193 - 101
22chain 'B' and (resid 102 through 120 )BB102 - 120102 - 120
33chain 'B' and (resid 121 through 133 )BB121 - 133121 - 133
44chain 'B' and (resid 134 through 151 )BB134 - 151134 - 151
55chain 'C' and (resid 3 through 12 )CC3 - 12
66chain 'D' and (resid 3 through 12 )DD3 - 12
77chain 'A' and (resid 2 through 17 )AA2 - 171 - 16
88chain 'A' and (resid 18 through 27 )AA18 - 2717 - 26
99chain 'A' and (resid 28 through 53 )AA28 - 5327 - 52
1010chain 'A' and (resid 54 through 82 )AA54 - 8253 - 81
1111chain 'A' and (resid 83 through 92 )AA83 - 9282 - 91
1212chain 'A' and (resid 93 through 101 )AA93 - 10192 - 100
1313chain 'A' and (resid 102 through 133 )AA102 - 133101 - 132
1414chain 'A' and (resid 134 through 160 )AA134 - 160133 - 159
1515chain 'B' and (resid 1 through 17 )BB1 - 171 - 17
1616chain 'B' and (resid 18 through 38 )BB18 - 3818 - 38
1717chain 'B' and (resid 39 through 70 )BB39 - 7039 - 70
1818chain 'B' and (resid 71 through 82 )BB71 - 8271 - 82
1919chain 'B' and (resid 83 through 92 )BB83 - 9283 - 92

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