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- PDB-6ltr: Crystal structure of Cas12i2 ternary complex with single Mg2+ bou... -

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Basic information

Entry
Database: PDB / ID: 6ltr
TitleCrystal structure of Cas12i2 ternary complex with single Mg2+ bound in catalytic pocket
Components
  • Cas12i2
  • DNA (35-MER)
  • DNA (5'-D(*GP*CP*CP*GP*CP*TP*TP*TP*CP*TP*TP*T)-3')
  • RNA (56-mer)
  • trans ssDNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas / Cas12i2 / Cas12i2 ternary complex / HYDROLASE / HYDROLASE-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesunidentified (others)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.51 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 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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*TP*T)-3')
C: DNA (35-MER)
E: trans ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,9507
Polymers155,8645
Non-polymers862
Water7,062392
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19830 Å2
ΔGint-129 kcal/mol
Surface area56780 Å2
MethodPISA
Unit cell
Length a, b, c (Å)93.256, 123.025, 280.420
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

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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*TP*T)-3')


Mass: 3899.531 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: 1762.208 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: 121411.398 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: 18051.766 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 394 molecules

#6: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C2H6O2
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 392 / 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: 54.5 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 7.5 / Details: 0.2 M Ammonium acetate, 20% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 0.9999 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: May 14, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9999 Å / Relative weight: 1
ReflectionResolution: 2.5→50 Å / Num. obs: 55232 / % possible obs: 99 % / Redundancy: 9.48 % / Biso Wilson estimate: 34.16 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.054 / Rpim(I) all: 0.022 / Rrim(I) all: 0.058 / Χ2: 0.97 / Net I/σ(I): 31.6
Reflection shellResolution: 2.5→2.545 Å / Redundancy: 5.1 % / Rmerge(I) obs: 0.353 / Mean I/σ(I) obs: 4.2 / Num. unique obs: 2586 / CC1/2: 0.936 / Rpim(I) all: 0.165 / Rrim(I) all: 0.391 / Χ2: 0.707 / % possible all: 94.8

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
PHENIX1.18.2_3874refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: SAD / Resolution: 2.51→46.74 Å / SU ML: 0.3055 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 22.5125
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.226 2746 5.02 %
Rwork0.1976 52005 -
obs0.199 54751 98.39 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 41.46 Å2
Refinement stepCycle: LAST / Resolution: 2.51→46.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8327 2076 5 392 10800
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.007510819
X-RAY DIFFRACTIONf_angle_d1.134215059
X-RAY DIFFRACTIONf_chiral_restr0.06881703
X-RAY DIFFRACTIONf_plane_restr0.00591571
X-RAY DIFFRACTIONf_dihedral_angle_d21.92454258
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.51-2.550.32791190.25412274X-RAY DIFFRACTION87.66
2.55-2.60.30561520.24572504X-RAY DIFFRACTION96.27
2.6-2.650.31751290.25092559X-RAY DIFFRACTION98.25
2.65-2.70.26271210.24622585X-RAY DIFFRACTION99.3
2.7-2.760.28941430.24452665X-RAY DIFFRACTION99.68
2.76-2.820.28441530.24882567X-RAY DIFFRACTION99.89
2.82-2.890.3011480.25162586X-RAY DIFFRACTION99.85
2.89-2.970.29851300.25012653X-RAY DIFFRACTION99.93
2.97-3.060.27751360.24392614X-RAY DIFFRACTION99.82
3.06-3.160.26761190.23192620X-RAY DIFFRACTION99.46
3.16-3.270.26071380.21872603X-RAY DIFFRACTION99.35
3.27-3.40.21871240.2052645X-RAY DIFFRACTION99.18
3.4-3.560.19331360.20252619X-RAY DIFFRACTION99.39
3.56-3.740.20481420.19892621X-RAY DIFFRACTION99.6
3.74-3.980.22151550.17322597X-RAY DIFFRACTION99.06
3.98-4.290.17891420.16792638X-RAY DIFFRACTION98.62
4.29-4.720.20271250.15192572X-RAY DIFFRACTION96.6
4.72-5.40.1741300.15682617X-RAY DIFFRACTION97.86
5.4-6.80.19091420.1812712X-RAY DIFFRACTION99.65
6.8-46.740.18071620.15652754X-RAY DIFFRACTION98.25

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