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- PDB-6je3: Crystal structure of Nme2Cas9 in complex with sgRNA and target DN... -

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Basic information

Entry
Database: PDB / ID: 6je3
TitleCrystal structure of Nme2Cas9 in complex with sgRNA and target DNA (AGGCCC PAM) with 5 nt overhang
Components
  • CRISPR-associated endonuclease Cas9
  • non-target DNA strand
  • sgRNA
  • target DNA strand
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / NmeCas9 / Nme2Cas9 / HYDROLASE-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesNeisseria meningitidis (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.931 Å
AuthorsSun, W. / Yang, J. / Cheng, Z. / Liu, C. / Wang, K. / Huang, X. / Wang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China31725008 China
CitationJournal: Mol.Cell / Year: 2019
Title: Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States.
Authors: Sun, W. / Yang, J. / Cheng, Z. / Amrani, N. / Liu, C. / Wang, K. / Ibraheim, R. / Edraki, A. / Huang, X. / Wang, M. / Wang, J. / Liu, L. / Sheng, G. / Yang, Y. / Lou, J. / Sontheimer, E.J. / Wang, Y.
History
DepositionFeb 3, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 8, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Nov 22, 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
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: sgRNA
C: target DNA strand
D: non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,8115
Polymers183,7494
Non-polymers621
Water27015
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19730 Å2
ΔGint-172 kcal/mol
Surface area62770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)166.388, 165.974, 178.760
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain target DNA strand


Mass: 10845.010 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain non-target DNA strand


Mass: 4931.230 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 CRISPR-associated endonuclease Cas9


Mass: 124913.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria) / Strain: DE10444 / Gene: cas9 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA


Mass: 43059.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 16 molecules

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


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


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY
Sequence detailsSequence of the protein has been deposited to NCBI with accession ID WP_002230835.1

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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: 3.36 Å3/Da / Density % sol: 63.37 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 5.2
Details: 10% PEG 6000, 1M LiCl, 0.1M citric acid pH 5.2, 0.01M Spermidine

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 1.06 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Dec 31, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.06 Å / Relative weight: 1
ReflectionResolution: 2.93→50 Å / Num. obs: 53110 / % possible obs: 99.9 % / Redundancy: 5.9 % / CC1/2: 0.932 / Rmerge(I) obs: 0.131 / Rpim(I) all: 0.058 / Rrim(I) all: 0.143 / Χ2: 0.917 / Net I/σ(I): 11.4
Reflection shellResolution: 2.93→2.98 Å / Redundancy: 5.1 % / Rmerge(I) obs: 0.954 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 2616 / CC1/2: 0.53 / Rpim(I) all: 0.457 / Rrim(I) all: 0.143 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX(1.14_3247: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6JDV

6jdv


Resolution: 2.931→41.597 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 25.9
RfactorNum. reflection% reflection
Rfree0.2561 1906 4.75 %
Rwork0.2265 --
obs0.2278 40154 75.34 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.931→41.597 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6328 3460 4 15 9807
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00410308
X-RAY DIFFRACTIONf_angle_d0.83214722
X-RAY DIFFRACTIONf_dihedral_angle_d14.1685767
X-RAY DIFFRACTIONf_chiral_restr0.0431763
X-RAY DIFFRACTIONf_plane_restr0.0051313
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.931-3.00430.3851610.3121025X-RAY DIFFRACTION29
3.0043-3.08550.3502730.27971320X-RAY DIFFRACTION37
3.0855-3.17620.3037900.26971631X-RAY DIFFRACTION46
3.1762-3.27870.30371000.23681866X-RAY DIFFRACTION52
3.2787-3.39590.2603910.22812187X-RAY DIFFRACTION60
3.3959-3.53180.29561330.23752403X-RAY DIFFRACTION67
3.5318-3.69240.25681510.22642740X-RAY DIFFRACTION76
3.6924-3.88690.26321580.22623135X-RAY DIFFRACTION87
3.8869-4.13030.25121570.21873545X-RAY DIFFRACTION98
4.1303-4.44880.25421920.21383611X-RAY DIFFRACTION100
4.4488-4.89590.23032000.20693632X-RAY DIFFRACTION100
4.8959-5.60290.26061780.21223651X-RAY DIFFRACTION100
5.6029-7.05350.23121460.24073711X-RAY DIFFRACTION100
7.0535-41.60120.23411760.22733791X-RAY DIFFRACTION99

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