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- PDB-6k4q: Crystal structure of xCas9 in complex with sgRNA and DNA (CGG PAM) -

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Basic information

Entry
Database: PDB / ID: 6k4q
TitleCrystal structure of xCas9 in complex with sgRNA and DNA (CGG PAM)
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (28-MER)
  • DNA (5'-D(*AP*AP*AP*CP*GP*GP*TP*AP*TP*TP*G)-3')
  • sgRNA
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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
PHOSPHATE ION / 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: 2.7 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 26, 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: sgRNA
C: DNA (28-MER)
D: DNA (5'-D(*AP*AP*AP*CP*GP*GP*TP*AP*TP*TP*G)-3')
B: CRISPR-associated endonuclease Cas9/Csn1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,49813
Polymers197,6434
Non-polymers8559
Water1,78399
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18860 Å2
ΔGint-162 kcal/mol
Surface area74660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)177.840, 69.669, 189.513
Angle α, β, γ (deg.)90.00, 109.35, 90.00
Int Tables number5
Space group name H-MC121

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Components

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DNA chain , 2 types, 2 molecules CD

#2: DNA chain DNA (28-MER)


Mass: 8568.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain DNA (5'-D(*AP*AP*AP*CP*GP*GP*TP*AP*TP*TP*G)-3')


Mass: 3710.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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RNA chain / Protein , 2 types, 2 molecules AB

#1: RNA chain sgRNA


Mass: 26788.984 Da / Num. of mol.: 1
Mutation: D10A, C80L, A262T, R324L, S409T, E480K, E543D, C574E, M694I, H840A, E1219V
Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158574.844 Da / Num. of mol.: 1
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

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Non-polymers , 2 types, 108 molecules

#5: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: PO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 99 / 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: 2.89 Å3/Da / Density % sol: 57.45 %
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: 0.9785 Å
DetectorType: AGILENT EOS CCD / Detector: CCD / Date: Sep 21, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9785 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. obs: 60255 / % possible obs: 99.8 % / Redundancy: 6.7 % / CC1/2: 0.995 / Rpim(I) all: 0.063 / Net I/σ(I): 11.6
Reflection shellResolution: 2.7→2.8 Å / Redundancy: 5.7 % / Num. unique obs: 5992 / CC1/2: 0.578 / Rpim(I) all: 0.506 / % possible all: 99.7

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Processing

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

4un3


Resolution: 2.7→49.38 Å / Cor.coef. Fo:Fc: 0.931 / Cor.coef. Fo:Fc free: 0.869 / SU B: 34.022 / SU ML: 0.308 / Cross valid method: THROUGHOUT / ESU R: 1.752 / ESU R Free: 0.381 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.27547 2885 5.1 %RANDOM
Rwork0.20335 ---
obs0.20702 54217 94.41 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 47.669 Å2
Baniso -1Baniso -2Baniso -3
1--0.81 Å20 Å2-0.75 Å2
2---1.3 Å20 Å2
3---2.11 Å2
Refinement stepCycle: 1 / Resolution: 2.7→49.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10539 2526 45 99 13209
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.01213589
X-RAY DIFFRACTIONr_bond_other_d0.0010.01811350
X-RAY DIFFRACTIONr_angle_refined_deg1.8321.55318897
X-RAY DIFFRACTIONr_angle_other_deg1.381.74526389
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.27351315
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.26922.487575
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.42151977
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.7931570
X-RAY DIFFRACTIONr_chiral_restr0.0890.21853
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0213311
X-RAY DIFFRACTIONr_gen_planes_other0.0020.022845
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_it1.8483.3995281
X-RAY DIFFRACTIONr_mcbond_other1.8473.3985279
X-RAY DIFFRACTIONr_mcangle_it3.0745.096589
X-RAY DIFFRACTIONr_mcangle_other3.0745.096589
X-RAY DIFFRACTIONr_scbond_it1.5683.1328308
X-RAY DIFFRACTIONr_scbond_other1.5013.1118273
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.5394.63512255
X-RAY DIFFRACTIONr_long_range_B_refined4.98734.91715657
X-RAY DIFFRACTIONr_long_range_B_other4.98634.91915655
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.703→2.773 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.418 119 -
Rwork0.353 2171 -
obs--51.94 %
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)
10.5193-0.2114-0.0590.4943-0.02930.22260.01630.08970.1114-0.03110.05960.0690.14660.0122-0.07580.1913-0.10270.01860.2237-0.03610.0976-2.094427.682633.8322
20.38510.05780.17480.16510.21250.3858-0.05230.0816-0.14850.12370.1027-0.00290.26630.0224-0.05040.3284-0.1280.02720.2465-0.01160.0603-6.831120.241651.9773
34.43040.8053-2.02244.7583-4.80725.4608-0.006-0.0088-1.02270.0305-0.1641-0.0985-0.19070.48180.17010.0987-0.20120.12630.4122-0.25680.3986-10.27558.717825.7775
40.63050.0455-0.05990.3474-0.03170.6037-0.0755-0.0173-0.07510.08090.0621-0.01780.11750.0090.01340.1439-0.04920.06030.0814-0.03450.044-5.368521.52552.309
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 81
2X-RAY DIFFRACTION2C1 - 28
3X-RAY DIFFRACTION3D2 - 12
4X-RAY DIFFRACTION4B3 - 1367

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