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- PDB-6m0x: Crystal structure of Streptococcus thermophilus Cas9 in complex w... -

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Basic information

Entry
Database: PDB / ID: 6m0x
TitleCrystal structure of Streptococcus thermophilus Cas9 in complex with AGGA PAM
Components
  • CRISPR-associated endonuclease Cas9 1
  • DNA (28-MER)
  • DNA (5'-D(*AP*AP*AP*GP*GP*AP*GP*C)-3')
  • RNA (71-MER)
KeywordsDNA BINDING PROTEIN / Cas9 / CRISPR
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / 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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...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 / 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 1
Similarity search - Component
Biological speciesStreptococcus thermophilus LMD-9 (bacteria)
Streptococcus thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.561 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 23, 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
A: CRISPR-associated endonuclease Cas9 1
B: RNA (71-MER)
C: DNA (28-MER)
D: DNA (5'-D(*AP*AP*AP*GP*GP*AP*GP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,46720
Polymers163,4964
Non-polymers1,97116
Water91951
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area25700 Å2
ΔGint-324 kcal/mol
Surface area54660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)322.165, 75.250, 70.008
Angle α, β, γ (deg.)90.000, 93.280, 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

#3: DNA chain DNA (28-MER)


Mass: 8481.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus (bacteria)
#4: DNA chain DNA (5'-D(*AP*AP*AP*GP*GP*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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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cas9 1 / Cas9**


Mass: 129646.898 Da / Num. of mol.: 1
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
#2: RNA chain RNA (71-MER)


Mass: 22882.627 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus (bacteria)

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Non-polymers , 3 types, 67 molecules

#5: Chemical
ChemComp-BA / BARIUM ION


Mass: 137.327 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Ba
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 51 / 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: 52.53 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / Details: KCL, BaCL2, 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 4, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978 Å / Relative weight: 1
ReflectionResolution: 2.56→50 Å / Num. obs: 54046 / % possible obs: 99.9 % / Redundancy: 6.7 % / Biso Wilson estimate: 49.21 Å2 / Rmerge(I) obs: 0.103 / Rpim(I) all: 0.043 / Rrim(I) all: 0.112 / Χ2: 0.953 / Net I/σ(I): 5.3 / Num. measured all: 360588
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.56-2.656.30.7253420.8630.310.7860.72399.7
2.65-2.766.50.58753590.9040.2470.6380.739100
2.76-2.886.50.43154050.9390.1830.470.776100
2.88-3.0470.30353600.9720.1230.3270.826100
3.04-3.236.90.18253690.9890.0750.1970.886100
3.23-3.476.50.13353920.9930.0560.1450.965100
3.47-3.8270.10354000.9940.0420.1121.079100
3.82-4.386.90.0854350.9960.0330.0861.213100
4.38-5.516.80.06654300.9960.0270.0711.203100
5.51-506.50.04955540.9980.0210.0531.07499.8

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Processing

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

5axw


Resolution: 2.561→48.896 Å / SU ML: 0.34 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 30.44 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2556 2749 5.09 %
Rwork0.2256 51261 -
obs0.2272 54010 99.71 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 182.97 Å2 / Biso mean: 60.1755 Å2 / Biso min: 28.57 Å2
Refinement stepCycle: final / Resolution: 2.561→48.896 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8229 2227 16 51 10523
Biso mean--105.26 44.76 -
Num. residues----1139
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00810865
X-RAY DIFFRACTIONf_angle_d0.90115146
X-RAY DIFFRACTIONf_chiral_restr0.0481738
X-RAY DIFFRACTIONf_plane_restr0.0061567
X-RAY DIFFRACTIONf_dihedral_angle_d19.8276284
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.5613-2.60550.36371130.3106250696
2.6055-2.65290.36111160.32122535100
2.6529-2.70390.34261160.30352613100
2.7039-2.75910.38831380.30412504100
2.7591-2.81910.39161520.29612552100
2.8191-2.88460.33091370.29852553100
2.8846-2.95680.37111460.30112527100
2.9568-3.03670.33541400.28452562100
3.0367-3.1260.29071190.27012578100
3.126-3.22690.28841280.25742554100
3.2269-3.34220.29411300.24192557100
3.3422-3.4760.31591560.23752542100
3.476-3.63420.23341510.22722561100
3.6342-3.82570.2391490.20882547100
3.8257-4.06530.21351460.19662574100
4.0653-4.3790.20731210.18662604100
4.379-4.81930.20661540.17742545100
4.8193-5.51590.2181620.18822571100
5.5159-6.94620.25971150.21772634100
6.9462-48.8960.20011600.1984264299
Refinement TLS params.Method: refined / Origin x: 36.3254 Å / Origin y: 12.6209 Å / Origin z: 145.946 Å
111213212223313233
T0.4134 Å20.0558 Å20.0209 Å2-0.4318 Å2-0.0167 Å2--0.4301 Å2
L1.1009 °20.1461 °20.3633 °2-0.6206 °20.007 °2--2.0746 °2
S-0.0602 Å °0.0438 Å °-0.0245 Å °-0.0829 Å °0.0397 Å °0.1593 Å °-0.1636 Å °-0.5032 Å °0.0207 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA4 - 1121
2X-RAY DIFFRACTION1allB0 - 70
3X-RAY DIFFRACTION1allC1 - 28
4X-RAY DIFFRACTION1allD1 - 8
5X-RAY DIFFRACTION1allF1 - 6
6X-RAY DIFFRACTION1allF7 - 11
7X-RAY DIFFRACTION1allF12 - 15
8X-RAY DIFFRACTION1allE1
9X-RAY DIFFRACTION1allE2
10X-RAY DIFFRACTION1allS1 - 51

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