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- PDB-6kc8: Crystal structure of WT Nme1Cas9 in complex with sgRNA and target... -

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Basic information

Entry
Database: PDB / ID: 6kc8
TitleCrystal structure of WT Nme1Cas9 in complex with sgRNA and target DNA (ATATGATT PAM) in post-cleavage 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*A)-3')
  • DNA (5'-D(P*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*GP*TP*GP*AP*CP*C)-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: 2.9 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(P*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*GP*TP*GP*AP*CP*C)-3')
D: DNA (5'-D(*AP*TP*AP*TP*GP*AP*TP*TP*TP*TP*A)-3')
P: DNA (5'-D(*TP*AP*AP*AP*AP*TP*CP*AP*TP*AP*TP*GP*TP*A)-3')


Theoretical massNumber of molelcules
Total (without water)181,9145
Polymers181,9145
Non-polymers00
Water1448
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23330 Å2
ΔGint-174 kcal/mol
Surface area67930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)131.604, 159.517, 117.963
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

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DNA chain , 3 types, 3 molecules CDP

#3: DNA chain DNA (5'-D(P*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*GP*TP*GP*AP*CP*C)-3')


Mass: 6504.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / 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)
#5: DNA chain DNA (5'-D(*TP*AP*AP*AP*AP*TP*CP*AP*TP*AP*TP*GP*TP*A)-3')


Mass: 4286.841 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain / Non-polymers , 3 types, 10 molecules AB

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 124700.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis 8013 (bacteria) / Strain: 8013 / 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)
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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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: 3.33 Å3/Da / Density % sol: 63.09 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 7.2
Details: 19% PEG 3350, 0.2M Na citrate, 0.1M Bis-Tris propane pH 7.2

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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: Jun 4, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.9→50 Å / Num. obs: 56256 / % possible obs: 99.8 % / Redundancy: 7 % / CC1/2: 0.986 / Rmerge(I) obs: 0.207 / Rpim(I) all: 0.079 / Rrim(I) all: 0.222 / Χ2: 0.932 / Net I/σ(I): 12.9
Reflection shellResolution: 2.9→2.95 Å / Redundancy: 5.6 % / Rmerge(I) obs: 0.969 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 2792 / CC1/2: 0.112 / Rpim(I) all: 0.409 / Rrim(I) all: 1.056 / Χ2: 0.831 / % possible all: 99.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6JDV

6jdv


Resolution: 2.9→49.35 Å / Cor.coef. Fo:Fc: 0.899 / Cor.coef. Fo:Fc free: 0.833 / SU B: 17.902 / SU ML: 0.323 / Cross valid method: FREE R-VALUE / ESU R Free: 0.416 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.25693 2422 5.2 %RANDOM
Rwork0.20278 ---
obs0.2056 44188 82.72 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 66.414 Å2
Baniso -1Baniso -2Baniso -3
1-1.4 Å20 Å20 Å2
2--0.14 Å2-0 Å2
3----1.54 Å2
Refinement stepCycle: 1 / Resolution: 2.9→49.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8095 3691 0 8 11794
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.01212365
X-RAY DIFFRACTIONr_bond_other_d0.0020.0189594
X-RAY DIFFRACTIONr_angle_refined_deg1.5521.50817514
X-RAY DIFFRACTIONr_angle_other_deg1.3751.85722312
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.51551030
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.22920.812468
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.643151451
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.5691582
X-RAY DIFFRACTIONr_chiral_restr0.080.21725
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0211347
X-RAY DIFFRACTIONr_gen_planes_other0.0020.022701
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it6.6597.354138
X-RAY DIFFRACTIONr_mcbond_other6.667.354137
X-RAY DIFFRACTIONr_mcangle_it10.54511.0155162
X-RAY DIFFRACTIONr_mcangle_other10.54411.0155163
X-RAY DIFFRACTIONr_scbond_it6.047.118227
X-RAY DIFFRACTIONr_scbond_other6.047.118228
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other9.66810.57612353
X-RAY DIFFRACTIONr_long_range_B_refined17.02151181
X-RAY DIFFRACTIONr_long_range_B_other17.02151181
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.9→2.965 Å
RfactorNum. reflection% reflection
Rfree0.306 55 -
Rwork0.333 1244 -
obs--31.38 %

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