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- PDB-6k3z: Crystal structure of dCas9 in complex with sgRNA and DNA (TGA PAM) -

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Basic information

Entry
Database: PDB / ID: 6k3z
TitleCrystal structure of dCas9 in complex with sgRNA and DNA (TGA PAM)
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (28-MER)
  • DNA (5'-D(*AP*AP*AP*TP*GP*AP*TP*AP*TP*TP*G)-3')
  • RNA (81-MER)
KeywordsHYDROLASE/DNA/RNA / endonuclease / HYDROLASE-DNA-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
Streptococcus pyogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsChen, W. / Zhang, H. / Zhang, Y. / Wang, Y. / Gan, J. / Ji, Q.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China91753127 China
CitationJournal: Plos Biol. / Year: 2019
Title: Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.
Authors: Chen, W. / Zhang, H. / Zhang, Y. / Wang, Y. / Gan, J. / Ji, Q.
History
DepositionMay 22, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Nov 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: RNA (81-MER)
B: CRISPR-associated endonuclease Cas9
C: DNA (28-MER)
D: DNA (5'-D(*AP*AP*AP*TP*GP*AP*TP*AP*TP*TP*G)-3')


Theoretical massNumber of molelcules
Total (without water)197,6554
Polymers197,6554
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18780 Å2
ΔGint-151 kcal/mol
Surface area76130 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.943, 69.058, 190.046
Angle α, β, γ (deg.)90.00, 110.21, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: RNA chain RNA (81-MER)


Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#2: Protein CRISPR-associated endonuclease Cas9 / SpCas9 / SpyCas9


Mass: 158588.781 Da / Num. of mol.: 1 / Mutation: H840A, D10A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: Cas9 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#3: DNA chain DNA (28-MER)


Mass: 8567.574 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: DNA chain DNA (5'-D(*AP*AP*AP*TP*GP*AP*TP*AP*TP*TP*G)-3')


Mass: 3709.466 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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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: 2.87 Å3/Da / Density % sol: 57.16 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.2M Ammonium phosphate dibasic, 20%(w/v) Polyethylene glycol 3,350

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Data collection

DiffractionMean temperature: 85 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97853 Å
DetectorType: AGILENT EOS CCD / Detector: CCD / Date: Mar 15, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 3.2→50 Å / Num. obs: 35665 / % possible obs: 98.3 % / Redundancy: 6.4 % / Rpim(I) all: 0.061 / Net I/σ(I): 12.1
Reflection shellResolution: 3.2→3.31 Å / Num. unique obs: 3541 / CC1/2: 0.748 / Rpim(I) all: 0.393 / % possible all: 97.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
HKL-3000data scaling
PHASERphasing
HKL-3000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4UN3

4un3


Resolution: 3.2→40.89 Å / Cor.coef. Fo:Fc: 0.908 / Cor.coef. Fo:Fc free: 0.827 / SU B: 63.976 / SU ML: 0.465 / Cross valid method: THROUGHOUT / ESU R Free: 0.623 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.27554 1564 4.9 %RANDOM
Rwork0.1969 ---
obs0.20075 30136 87.03 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parameters
Baniso -1Baniso -2Baniso -3
1--1.66 Å20 Å22.37 Å2
2---7.1 Å2-0 Å2
3---5.52 Å2
Refinement stepCycle: 1 / Resolution: 3.2→40.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10758 2526 0 0 13284
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.01213778
X-RAY DIFFRACTIONr_bond_other_d0.0020.01811723
X-RAY DIFFRACTIONr_angle_refined_deg1.7661.55619101
X-RAY DIFFRACTIONr_angle_other_deg1.2771.74127357
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.70751308
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.94322.63616
X-RAY DIFFRACTIONr_dihedral_angle_3_deg23.137152144
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.2161575
X-RAY DIFFRACTIONr_chiral_restr0.090.21847
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0213421
X-RAY DIFFRACTIONr_gen_planes_other0.0060.022862
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_it2.7325.4485253
X-RAY DIFFRACTIONr_mcbond_other2.735.4485252
X-RAY DIFFRACTIONr_mcangle_it4.6718.1656554
X-RAY DIFFRACTIONr_mcangle_other4.6718.1656555
X-RAY DIFFRACTIONr_scbond_it2.3315.3928525
X-RAY DIFFRACTIONr_scbond_other2.3315.3918524
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.0387.9912548
X-RAY DIFFRACTIONr_long_range_B_refined6.92358.66916210
X-RAY DIFFRACTIONr_long_range_B_other6.92358.67116211
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 3.195→3.278 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.333 39 -
Rwork0.273 1070 -
obs--41.43 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.1307-0.19980.02950.3445-0.06110.0725-0.01970.0047-0.0275-0.02990.08750.0650.0690.0675-0.06790.236-0.1504-0.03070.547-0.06160.068686.331962.468634.1724
20.2182-0.0276-0.00350.1345-0.13360.3472-0.08490.0969-0.01260.07980.0992-0.00050.0320.0029-0.01430.2213-0.06950.02750.3999-0.06410.013183.694456.402653.0289
30.2413-0.0995-0.07380.08410.05220.0361-0.23540.043-0.13730.0880.15960.05870.0610.06860.07580.3323-0.14290.1340.5385-0.00660.085281.495155.482952.2748
41.6361.699-0.19841.9758-0.10460.0728-0.2332-0.0928-0.3659-0.29740.0644-0.12610.00330.08380.16890.1589-0.14950.05120.44510.08110.455178.348843.511427.0924
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 81
2X-RAY DIFFRACTION2B4 - 1364
3X-RAY DIFFRACTION3C1 - 28
4X-RAY DIFFRACTION4D2 - 12

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