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- PDB-6m0v: Crsytal structure of streptococcus thermophilus Cas9 in complex w... -

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Basic information

Entry
Database: PDB / ID: 6m0v
TitleCrsytal structure of streptococcus thermophilus Cas9 in complex with the GGAA PAM
Components
  • CRISPR-associated endonuclease Cas9 1
  • DNA (28-MER)
  • DNA (5'-D(*AP*AP*GP*GP*AP*AP*GP*C)-3')
  • RNA (71-MER)
KeywordsHYDROLASE/DNA/RNA / CRISPR / HYDROLASE-DNA-RNA 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
Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / 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) / CRISPR-associated endonuclease Cas9 1
Similarity search - Component
Biological speciesStreptococcus thermophilus LMD-9 (bacteria)
Streptococcus thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsZhang, Y. / Zhang, H. / Xu, X. / Wang, Y. / Chen, W. / Wang, Y. / Wu, Z. / Tang, N. / Wang, Y. / Zhao, S. ...Zhang, Y. / Zhang, H. / Xu, X. / Wang, Y. / Chen, W. / Wang, Y. / Wu, Z. / Tang, N. / Wang, Y. / Zhao, S. / Gan, J. / Ji, Q.
CitationJournal: Nat Catal / Year: 2020
Title: Catalytic-state structure and engineering of Streptococcus thermophilus Cas9
Authors: Zhang, Y. / Zhang, H. / Xu, X. / Wang, Y. / Chen, W. / Wang, Y. / Wu, Z. / Tang, N. / Wang, Y. / Zhao, S. / Gan, J. / Ji, Q.
History
DepositionFeb 22, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 9, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 29, 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
B: RNA (71-MER)
C: DNA (28-MER)
D: DNA (5'-D(*AP*AP*GP*GP*AP*AP*GP*C)-3')
A: CRISPR-associated endonuclease Cas9 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,89315
Polymers163,4964
Non-polymers1,39811
Water1629
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24710 Å2
ΔGint-279 kcal/mol
Surface area54660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)321.839, 75.078, 70.109
Angle α, β, γ (deg.)90.000, 93.210, 90.000
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: 8481.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus (bacteria)
#3: DNA chain DNA (5'-D(*AP*AP*GP*GP*AP*AP*GP*C)-3')


Mass: 2484.669 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus (bacteria)

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RNA chain / Protein , 2 types, 2 molecules BA

#1: RNA chain RNA (71-MER)


Mass: 22882.627 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus (bacteria)
#4: Protein CRISPR-associated endonuclease Cas9 1 / Cas9**


Mass: 129646.898 Da / Num. of mol.: 1 / Mutation: H599A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus LMD-9 (bacteria)
Strain: LMD-9 / Gene: cas9-1, csn1, STER_0709 / Production host: Escherichia coli (E. coli)
References: UniProt: Q03LF7, Hydrolases; Acting on ester bonds

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Non-polymers , 3 types, 20 molecules

#5: Chemical
ChemComp-BA / BARIUM ION


Mass: 137.327 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Ba
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 2.59 Å3/Da / Density % sol: 52.44 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / Details: KCL, MgCl2, MPD, MgCl2

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Data collection

DiffractionMean temperature: 200 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.978 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 5, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978 Å / Relative weight: 1
ReflectionResolution: 3→50 Å / Num. obs: 31477 / % possible obs: 99.8 % / Redundancy: 6.6 % / Biso Wilson estimate: 57.43 Å2 / CC1/2: 0.8 / Net I/σ(I): 11.6
Reflection shellResolution: 3→3.11 Å / Num. unique obs: 1824 / CC1/2: 0.8

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHENIX1.10.1_2155refinement
PDB_EXTRACT3.25data extraction
HKL-3000data reduction
HKL-3000phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5axw

5axw


Resolution: 3→48.822 Å / SU ML: 0.38 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 26.71
Details: SF FILE CONTAINS FRIEDEL PAIRS UNDER I/F_MINUS AND I/F_PLUS COLUMNS.
RfactorNum. reflection% reflection
Rfree0.2566 1588 5.05 %
Rwork0.2134 --
obs0.2156 31457 93.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 245.41 Å2 / Biso mean: 53.4285 Å2 / Biso min: 16.1 Å2
Refinement stepCycle: final / Resolution: 3→48.822 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8235 2227 11 9 10482
Biso mean--105.32 32.5 -
Num. residues----1137
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00310882
X-RAY DIFFRACTIONf_angle_d0.64715164
X-RAY DIFFRACTIONf_chiral_restr0.0391739
X-RAY DIFFRACTIONf_plane_restr0.0041570
X-RAY DIFFRACTIONf_dihedral_angle_d19.2856296
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.0004-3.09720.3528780.2751154554
3.0972-3.20790.37421030.2634221476
3.2079-3.33630.28361620.2509275796
3.3363-3.48810.34051450.24312868100
3.4881-3.6720.27631770.23992915100
3.672-3.9020.26211360.21142917100
3.902-4.20310.27141770.19372852100
4.2031-4.62580.23221670.18142913100
4.6258-5.29440.21251150.17762962100
5.2944-6.66770.22571570.21352932100
6.6677-48.8220.22751710.2181299499

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