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

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Basic information

Entry
Database: PDB / ID: 6jdv
TitleCrystal structure of Nme1Cas9 in complex with sgRNA and target DNA (ATATGATT PAM) in catalytic state
Components
  • CRISPR-associated endonuclease Cas9
  • non-target DNA strand
  • sgRNA
  • target DNA strand
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 serogroup C (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 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 2, 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 ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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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)185,8206
Polymers185,7344
Non-polymers862
Water18010
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24390 Å2
ΔGint-193 kcal/mol
Surface area67360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)130.541, 158.340, 118.253
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 , 2 types, 2 molecules CD

#3: DNA chain target DNA strand


Mass: 10836.049 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain non-target DNA strand


Mass: 3362.232 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: 124633.898 Da / Num. of mol.: 1 / Mutation: H588A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis serogroup C (strain 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: 46901.605 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 12 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


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


Mass: 18.015 Da / Num. of mol.: 10 / 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: 3.29 Å3/Da / Density % sol: 62.61 %
Crystal growTemperature: 289 K / Method: evaporation / pH: 8 / Details: Na citrate, 0.1M HEPES pH 8.0, 18.5% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9786 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jan 15, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9786 Å / Relative weight: 1
ReflectionResolution: 3.1→50 Å / Num. obs: 45911 / % possible obs: 99.8 % / Redundancy: 7.4 % / CC1/2: 0.987 / Rmerge(I) obs: 0.179 / Rpim(I) all: 0.07 / Rrim(I) all: 0.193 / Χ2: 0.978 / Net I/σ(I): 11.7
Reflection shellResolution: 3.1→3.15 Å / Redundancy: 7.7 % / Rmerge(I) obs: 0.888 / Mean I/σ(I) obs: 2.3 / Num. unique obs: 2234 / CC1/2: 0.904 / Rpim(I) all: 0.336 / Rrim(I) all: 0.951 / Χ2: 0.925 / % 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: 5X2G

5x2g


Resolution: 3.1→41.958 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.21 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2314 2003 4.86 %
Rwork0.2123 --
obs0.2132 41232 91.32 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.1→41.958 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8226 3964 5 10 12205
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01312833
X-RAY DIFFRACTIONf_angle_d1.20918203
X-RAY DIFFRACTIONf_dihedral_angle_d20.1277394
X-RAY DIFFRACTIONf_chiral_restr0.0812128
X-RAY DIFFRACTIONf_plane_restr0.011673
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.17750.3236840.28011429X-RAY DIFFRACTION48
3.1775-3.26340.3091990.26561885X-RAY DIFFRACTION62
3.2634-3.35940.34641310.27662414X-RAY DIFFRACTION80
3.3594-3.46780.29541300.28232920X-RAY DIFFRACTION95
3.4678-3.59170.27871470.26633014X-RAY DIFFRACTION99
3.5917-3.73540.27641420.24053017X-RAY DIFFRACTION100
3.7354-3.90530.25821430.22723054X-RAY DIFFRACTION100
3.9053-4.1110.22921610.21093044X-RAY DIFFRACTION100
4.111-4.36830.2041360.19193082X-RAY DIFFRACTION100
4.3683-4.70520.20011850.18873026X-RAY DIFFRACTION100
4.7052-5.17790.20911470.18393090X-RAY DIFFRACTION100
5.1779-5.92540.20361800.19353070X-RAY DIFFRACTION100
5.9254-7.45850.2231550.20553096X-RAY DIFFRACTION99
7.4585-41.9620.20061630.18553088X-RAY DIFFRACTION95

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