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

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Basic information

Entry
Database: PDB / ID: 6jfu
TitleCrystal structure of Nme2Cas9 in complex with sgRNA and target DNA (AGGCCC PAM)
Components
  • CRISPR-associated endonuclease Cas9
  • non-target strand
  • sgRNA
  • target-strand DNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / NmeCas9 / Nme2Cas9 / hydrolase / 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: 3.2 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 12, 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

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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-strand DNA
D: non-target strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,3006
Polymers182,1764
Non-polymers1242
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19390 Å2
ΔGint-152 kcal/mol
Surface area62430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)153.477, 166.933, 179.790
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#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)
#3: DNA chain target-strand DNA


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


Mass: 3358.211 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
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: 3.16 Å3/Da / Density % sol: 61.08 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 5.6
Details: 10% PEG 6000, 1.2M LiCl, 0.1M citrate acid pH 5.6, 4% 1,1,1,3,3,3-Hexafluoro-2-propanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.97916 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Dec 6, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97916 Å / Relative weight: 1
ReflectionResolution: 3.2→50 Å / Num. obs: 38356 / % possible obs: 99.7 % / Redundancy: 7.3 % / CC1/2: 1 / Rmerge(I) obs: 0.243 / Rpim(I) all: 0.093 / Rrim(I) all: 0.261 / Χ2: 0.956 / Net I/σ(I): 10.2
Reflection shellResolution: 3.2→3.26 Å / Redundancy: 5.7 % / Rmerge(I) obs: 0.739 / Mean I/σ(I) obs: 2.8 / Num. unique obs: 1879 / CC1/2: 0.837 / Rpim(I) all: 0.323 / Rrim(I) all: 0.81 / Χ2: 0.908 / % possible all: 99.6

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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: 3.2→48.681 Å / SU ML: 0.39 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 28.72
RfactorNum. reflection% reflection
Rfree0.2707 1528 4.79 %
Rwork0.2535 --
obs0.2543 31895 83.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.2→48.681 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6312 3156 8 0 9476
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0039958
X-RAY DIFFRACTIONf_angle_d0.64914201
X-RAY DIFFRACTIONf_dihedral_angle_d14.1485583
X-RAY DIFFRACTIONf_chiral_restr0.0371718
X-RAY DIFFRACTIONf_plane_restr0.0041305
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2001-3.30330.3119690.26471566X-RAY DIFFRACTION48
3.3033-3.42140.2778860.25771875X-RAY DIFFRACTION57
3.4214-3.55830.302880.25772135X-RAY DIFFRACTION65
3.5583-3.72020.28471290.2572355X-RAY DIFFRACTION72
3.7202-3.91630.24761350.26822712X-RAY DIFFRACTION82
3.9163-4.16150.31141470.2673068X-RAY DIFFRACTION93
4.1615-4.48260.2841630.25923259X-RAY DIFFRACTION99
4.4826-4.93330.28531700.24323296X-RAY DIFFRACTION99
4.9333-5.64630.27711710.24753309X-RAY DIFFRACTION100
5.6463-7.11010.2721920.2733340X-RAY DIFFRACTION100
7.1101-48.68670.22931780.2343452X-RAY DIFFRACTION99

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