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- PDB-6ltu: Crystal structure of Cas12i2 ternary complex with double Mg2+ bou... -

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Basic information

Entry
Database: PDB / ID: 6ltu
TitleCrystal structure of Cas12i2 ternary complex with double Mg2+ bound in catalytic pocket
Components
  • Cas12i2
  • DNA (35-MER)
  • DNA (5'-D(*GP*CP*CP*GP*CP*TP*TP*TP*CP*TP*T)-3')
  • RNA (56-mer)
  • trans ssDNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas / Cas12i2 / Cas12i2 ternary complex / HYDROLASE / HYDROLASE-RNA-DNA complex / active RuvC state / wild type Cas12i2 / wt Cas12i2
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesunidentified (others)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.57 Å
AuthorsHuang, X. / Sun, W. / Cheng, Z. / Chen, M. / Li, X. / Wang, J. / Sheng, G. / Gong, W. / Wang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31630015 China
National Natural Science Foundation of China (NSFC)31725008 China
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2.
Authors: Huang, X. / Sun, W. / Cheng, Z. / Chen, M. / Li, X. / Wang, J. / Sheng, G. / Gong, W. / Wang, Y.
History
DepositionJan 23, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 28, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 4, 2020Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 29, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas12i2
B: RNA (56-mer)
D: DNA (5'-D(*GP*CP*CP*GP*CP*TP*TP*TP*CP*TP*T)-3')
C: DNA (35-MER)
E: trans ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,0679
Polymers155,9325
Non-polymers1354
Water5,855325
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)94.329, 123.575, 281.218
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2
Components on special symmetry positions
IDModelComponents
11B-220-

HOH

21B-246-

HOH

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Components

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DNA chain , 3 types, 3 molecules DCE

#3: DNA chain DNA (5'-D(*GP*CP*CP*GP*CP*TP*TP*TP*CP*TP*T)-3')


Mass: 3595.338 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (35-MER)


Mass: 10738.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain trans ssDNA


Mass: 1464.013 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein Cas12i2


Mass: 121469.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: RNA chain RNA (56-mer)


Mass: 18664.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 329 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 325 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Sequence details(1) Chain A contains the expression tag SER (0). (2) The author does not know the exact sequence of ...(1) Chain A contains the expression tag SER (0). (2) The author does not know the exact sequence of chain E. This short DNA chain is the substrate of Cas12i2 which is a DNA nuclease and it is bound in the catalytic pocket of the protein. The author built the model manually based on the Fo-Fc map and then refined the model. Due to the limited resolution, the author couldn't distinguish between DT and DC, DA and DG.

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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: 2.7 Å3/Da / Density % sol: 54.42 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 7.2 / Details: 0.2 M NaCl, 0.1 M Tris pH 7.2, 18% PEG 6000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 12, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.57→50 Å / Num. obs: 52840 / % possible obs: 100 % / Redundancy: 6.8 % / Biso Wilson estimate: 41.69 Å2 / CC1/2: 0.989 / Rmerge(I) obs: 0.16 / Rpim(I) all: 0.066 / Rrim(I) all: 0.174 / Χ2: 0.946 / Net I/σ(I): 12.7
Reflection shellResolution: 2.57→2.61 Å / Redundancy: 7.2 % / Rmerge(I) obs: 0.985 / Mean I/σ(I) obs: 2.2 / Num. unique obs: 2646 / CC1/2: 0.715 / Rpim(I) all: 0.395 / Rrim(I) all: 1.063 / Χ2: 0.847 / % possible all: 99.3

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6LTR

6ltr


Resolution: 2.57→46.87 Å / SU ML: 0.2979 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.1049
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2288 2504 4.9 %
Rwork0.202 48604 -
obs0.2033 51108 96.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 49.98 Å2
Refinement stepCycle: LAST / Resolution: 2.57→46.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8306 2000 67 325 10698
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.007210785
X-RAY DIFFRACTIONf_angle_d1.06215008
X-RAY DIFFRACTIONf_chiral_restr0.06661699
X-RAY DIFFRACTIONf_plane_restr0.00571574
X-RAY DIFFRACTIONf_dihedral_angle_d20.78132202
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.57-2.620.28921160.26722346X-RAY DIFFRACTION84.78
2.62-2.670.34641260.27072422X-RAY DIFFRACTION87.95
2.67-2.730.28921230.26582524X-RAY DIFFRACTION92.91
2.73-2.790.31291430.25772678X-RAY DIFFRACTION97.01
2.79-2.860.25981400.25352753X-RAY DIFFRACTION98.87
2.86-2.940.27541430.25022731X-RAY DIFFRACTION99.62
2.94-3.030.2621660.24362720X-RAY DIFFRACTION99.69
3.03-3.130.24371600.23672744X-RAY DIFFRACTION99.79
3.13-3.240.26831330.23942798X-RAY DIFFRACTION99.93
3.24-3.370.21721240.21762769X-RAY DIFFRACTION99.59
3.37-3.520.24921330.21282765X-RAY DIFFRACTION99.83
3.52-3.710.22521290.20652770X-RAY DIFFRACTION99.55
3.71-3.940.23911420.19932738X-RAY DIFFRACTION98.36
3.94-4.240.22621330.17052709X-RAY DIFFRACTION97.26
4.24-4.670.17451410.15892693X-RAY DIFFRACTION96.23
4.67-5.340.19971450.15992735X-RAY DIFFRACTION97.3
5.34-6.730.1971600.18062814X-RAY DIFFRACTION99.3
6.73-46.870.19341470.1732895X-RAY DIFFRACTION97.53

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