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- PDB-6kc7: Crystal structure of Nme1Cas9 in complex with sgRNA and target DN... -

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Basic information

Entry
Database: PDB / ID: 6kc7
TitleCrystal structure of Nme1Cas9 in complex with sgRNA and target DNA (ATATGATT PAM) in seed-base paring state
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (5'-D(*AP*TP*AP*TP*GP*AP*TP*TP*TP*TP*A)-3')
  • DNA (5'-D(*TP*AP*AP*AP*AP*TP*CP*AP*TP*AP*TP*GP*TP*AP*AP*AP*GP*TP*T)-3')
  • sgRNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / NmeCas9 / Nme1Cas9 / hydrolase / ternary complex / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesNeisseria meningitidis 8013 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 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
DepositionJun 27, 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: DNA (5'-D(*TP*AP*AP*AP*AP*TP*CP*AP*TP*AP*TP*GP*TP*AP*AP*AP*GP*TP*T)-3')
D: DNA (5'-D(*AP*TP*AP*TP*GP*AP*TP*TP*TP*TP*A)-3')


Theoretical massNumber of molelcules
Total (without water)176,9064
Polymers176,9064
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15700 Å2
ΔGint-134 kcal/mol
Surface area65510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)235.579, 72.513, 115.154
Angle α, β, γ (deg.)90.00, 104.67, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 124633.898 Da / Num. of mol.: 1 / Mutation: H588A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis 8013 (bacteria) / Gene: cas9, NMV_1993 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: C9X1G5, 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 DNA (5'-D(*TP*AP*AP*AP*AP*TP*CP*AP*TP*AP*TP*GP*TP*AP*AP*AP*GP*TP*T)-3')


Mass: 5850.847 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: TS8 target DNA / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(*AP*TP*AP*TP*GP*AP*TP*TP*TP*TP*A)-3')


Mass: 3362.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: non-target DNA strand / Source: (synth.) synthetic construct (others)

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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: 2.69 Å3/Da / Density % sol: 54.26 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 8
Details: 0.2M Na nitrate, 0.1M Bis-Tris propane pH 8.0, 20% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.97919 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 1, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97919 Å / Relative weight: 1
ReflectionResolution: 3.3→50 Å / Num. obs: 28597 / % possible obs: 99.9 % / Redundancy: 4.1 % / CC1/2: 0.991 / Rmerge(I) obs: 0.095 / Rpim(I) all: 0.053 / Rrim(I) all: 0.11 / Χ2: 0.922 / Net I/σ(I): 15.9
Reflection shellResolution: 3.3→3.42 Å / Redundancy: 3.9 % / Rmerge(I) obs: 0.906 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 2824 / CC1/2: 0.991 / Rpim(I) all: 0.516 / Rrim(I) all: 1.048 / Χ2: 1 / % 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: 6JDQ

6jdq


Resolution: 3.3→46.2 Å / SU ML: 0.44 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 29.3
RfactorNum. reflection% reflection
Rfree0.2745 1251 5.06 %
Rwork0.2526 --
obs0.2536 24709 86.01 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.3→46.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7540 2962 0 0 10502
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0110993
X-RAY DIFFRACTIONf_angle_d1.1115503
X-RAY DIFFRACTIONf_dihedral_angle_d23.9884483
X-RAY DIFFRACTIONf_chiral_restr0.0821810
X-RAY DIFFRACTIONf_plane_restr0.0071496
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3-3.42880.3857470.3094898X-RAY DIFFRACTION30
3.4288-3.58480.3158900.29871783X-RAY DIFFRACTION59
3.5848-3.77370.32711260.27452577X-RAY DIFFRACTION85
3.7737-4.010.29481520.26973007X-RAY DIFFRACTION99
4.01-4.31940.25481520.24072998X-RAY DIFFRACTION100
4.3194-4.75370.2711610.22993016X-RAY DIFFRACTION100
4.7537-5.44060.26991760.22763029X-RAY DIFFRACTION100
5.4406-6.85110.25961710.25413032X-RAY DIFFRACTION100
6.8511-46.20.24941760.25073118X-RAY DIFFRACTION100

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