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- PDB-8y0d: Crystal structure of SauCas9 in complex with sgRNA and 20nt ssDNA... -

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Basic information

Entry
Database: PDB / ID: 8y0d
TitleCrystal structure of SauCas9 in complex with sgRNA and 20nt ssDNA target
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (5'-D(P*GP*TP*AP*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*C)-3')
  • RNA (73-MER)
KeywordsRNA BINDING PROTEIN / CRISPR / Cas12a / crRNA / 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
: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / 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
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.92 Å
AuthorsChen, Y. / Chen, J. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32022047 China
CitationJournal: Nucleic Acids Res. / Year: 2025
Title: DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.
Authors: Chen, J. / Lin, X. / Xiang, W. / Chen, Y. / Zhao, Y. / Huang, L. / Liu, L.
History
DepositionJan 22, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RNA (73-MER)
C: DNA (5'-D(P*GP*TP*AP*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*C)-3')
A: CRISPR-associated endonuclease Cas9


Theoretical massNumber of molelcules
Total (without water)153,8943
Polymers153,8943
Non-polymers00
Water905
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12980 Å2
ΔGint-115 kcal/mol
Surface area61400 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.962, 145.567, 97.528
Angle α, β, γ (deg.)90.000, 93.986, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: RNA chain RNA (73-MER)


Mass: 23634.973 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria)
#2: DNA chain DNA (5'-D(P*GP*TP*AP*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*C)-3')


Mass: 6175.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria)
#3: Protein CRISPR-associated endonuclease Cas9 / SaCas9


Mass: 124083.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: cas9 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: J7RUA5, Hydrolases; Acting on ester bonds
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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: 3.17 Å3/Da / Density % sol: 61.24 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Sodium chloride, 0.1 M Sodium HEPES, pH 7.0, 11% PEG 8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.97915 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 16, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3.9→50 Å / Num. obs: 17461 / % possible obs: 98.9 % / Redundancy: 4.5 % / Biso Wilson estimate: 6 Å2 / Rpim(I) all: 0.063 / Net I/σ(I): 9.67
Reflection shellResolution: 3.9→3.97 Å / Redundancy: 3.3 % / Num. unique obs: 827 / Rpim(I) all: 0.432 / % possible all: 97.3

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-30007.21data scaling
HKL-30007.21data reduction
Coot0.7.2.1model building
PHENIX1.17.1_3660phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5AXW

5axw


Resolution: 3.92→48.65 Å / SU ML: 0.683 / Cross valid method: FREE R-VALUE / σ(F): 1.42 / Phase error: 29.7947
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3176 1325 9.97 %
Rwork0.2859 11962 -
obs0.289 13287 76.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 40.58 Å2
Refinement stepCycle: LAST / Resolution: 3.92→48.65 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8093 1925 0 5 10023
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.005710385
X-RAY DIFFRACTIONf_angle_d0.802314460
X-RAY DIFFRACTIONf_chiral_restr0.04841684
X-RAY DIFFRACTIONf_plane_restr0.00431522
X-RAY DIFFRACTIONf_dihedral_angle_d23.50394141
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.92-4.080.194140.2569107X-RAY DIFFRACTION6.28
4.08-4.270.3052690.2549700X-RAY DIFFRACTION40.39
4.27-4.490.29821350.26021182X-RAY DIFFRACTION70.17
4.49-4.770.29661600.27571499X-RAY DIFFRACTION86.5
4.77-5.140.30391800.29161618X-RAY DIFFRACTION93.84
5.14-5.660.36161860.3081686X-RAY DIFFRACTION97.65
5.66-6.470.38811880.33661707X-RAY DIFFRACTION99.01
6.47-8.150.35442000.32371719X-RAY DIFFRACTION99.74
8.15-48.650.28671930.26151744X-RAY DIFFRACTION98.88

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