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- PDB-8kal: Crystal structure of SpyCas9 in complex with sgRNA and 17nt target DNA -

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Basic information

Entry
Database: PDB / ID: 8kal
TitleCrystal structure of SpyCas9 in complex with sgRNA and 17nt target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (25-MER)
  • DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
  • RNA (98-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Nuclease / RNA BINDING PROTEIN-RNA-DNA 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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...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 / 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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.16 Å
AuthorsChen, Y. / Chen, J. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32022047 China
CitationJournal: Nat.Biotechnol. / Year: 2024
Title: Trans-nuclease activity of Cas9 activated by DNA or RNA target binding.
Authors: Chen, J. / Chen, Y. / Huang, L. / Lin, X. / Chen, H. / Xiang, W. / Liu, L.
History
DepositionAug 3, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (98-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (25-MER)
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
E: RNA (98-MER)
G: CRISPR-associated endonuclease Cas9/Csn1
H: DNA (25-MER)
J: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)402,60210
Polymers402,4098
Non-polymers1922
Water1,13563
1
A: RNA (98-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (25-MER)
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,3015
Polymers201,2054
Non-polymers961
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18810 Å2
ΔGint-168 kcal/mol
Surface area80540 Å2
MethodPISA
2
E: RNA (98-MER)
G: CRISPR-associated endonuclease Cas9/Csn1
H: DNA (25-MER)
J: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,3015
Polymers201,2054
Non-polymers961
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19000 Å2
ΔGint-155 kcal/mol
Surface area79440 Å2
MethodPISA
Unit cell
Length a, b, c (Å)362.049, 70.956, 200.100
Angle α, β, γ (deg.)90.000, 101.524, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
12
22
13
23
14
24

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111CCCCchain 'A'AA5 - 985 - 98
221CCCC(chain 'E' and resid 5 through 98)EE5 - 985 - 98
112LYSLYSARGARG(chain 'B' and (resid 4 through 1241 or (resid 1242...BB4 - 7654 - 765
122ASNASNASPASP(chain 'B' and (resid 4 through 1241 or (resid 1242...BB776 - 1012776 - 1012
132GLYGLYILEILE(chain 'B' and (resid 4 through 1241 or (resid 1242...BB1030 - 10501030 - 1050
142LYSLYSGLNGLN(chain 'B' and (resid 4 through 1241 or (resid 1242...BB1059 - 13641059 - 1364
252LYSLYSARGARG(chain 'G' and (resid 4 through 64 or (resid 65...GF4 - 7654 - 765
262ASNASNASPASP(chain 'G' and (resid 4 through 64 or (resid 65...GF776 - 1012776 - 1012
272GLYGLYILEILE(chain 'G' and (resid 4 through 64 or (resid 65...GF1030 - 10501030 - 1050
282LYSLYSGLNGLN(chain 'G' and (resid 4 through 64 or (resid 65...GF1059 - 13641059 - 1364
113DCDCDGDGchain 'C'CC1 - 251 - 25
223DCDCDGDGchain 'H'HG1 - 251 - 25
114DTDTDGDGchain 'D'DD2 - 121 - 11
224DTDTDGDGchain 'J'JH2 - 121 - 11

NCS ensembles :
ID
1
2
3
4

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Components

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DNA chain , 2 types, 4 molecules CHDJ

#3: DNA chain DNA (25-MER)


Mass: 7610.952 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: TS
Source: (synth.) Streptococcus pyogenes serotype M1 (bacteria)
#4: DNA chain DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')


Mass: 3394.230 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: NTS
Source: (synth.) Streptococcus pyogenes serotype M1 (bacteria)

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RNA chain / Protein , 2 types, 4 molecules AEBG

#1: RNA chain RNA (98-MER)


Mass: 31610.740 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Production host: Escherichia coli DH5[alpha] (bacteria)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158588.781 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 2 types, 65 molecules

#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 63 / 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: 3.22 Å3/Da / Density % sol: 61.86 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Ammonium sulfate, 0.1 M MES pH 6.0, 12.625% PEG8000

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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: Mar 15, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3.15→50.17 Å / Num. obs: 83075 / % possible obs: 99.1 % / Redundancy: 4.5 % / Biso Wilson estimate: 58.11 Å2 / Rpim(I) all: 0.187 / Net I/σ(I): 3
Reflection shellResolution: 3.15→3.2 Å / Num. unique obs: 4098 / Rpim(I) all: 0.54 / % possible all: 98

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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 / Resolution: 3.16→50.17 Å / SU ML: 0.4583 / Cross valid method: FREE R-VALUE / σ(F): 1.99 / Phase error: 31.6112
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2884 2796 4.94 %
Rwork0.2508 53831 -
obs0.2526 56627 65.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 59.6 Å2
Refinement stepCycle: LAST / Resolution: 3.16→50.17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21643 5498 10 63 27214
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011728188
X-RAY DIFFRACTIONf_angle_d1.556739167
X-RAY DIFFRACTIONf_chiral_restr0.17724487
X-RAY DIFFRACTIONf_plane_restr0.00614062
X-RAY DIFFRACTIONf_dihedral_angle_d22.993911469
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.16-3.210.484270.2639205X-RAY DIFFRACTION4.85
3.22-3.270.3263130.2929311X-RAY DIFFRACTION7.75
3.27-3.340.3373240.3074422X-RAY DIFFRACTION10.44
3.34-3.40.3195350.3235518X-RAY DIFFRACTION12.98
3.4-3.480.3239490.3205773X-RAY DIFFRACTION19.27
3.48-3.560.3118700.32291125X-RAY DIFFRACTION27.67
3.56-3.650.3173770.30671566X-RAY DIFFRACTION38.96
3.65-3.750.29971200.28472359X-RAY DIFFRACTION57.15
3.75-3.860.27051510.26422866X-RAY DIFFRACTION71.22
3.86-3.980.30891640.2693348X-RAY DIFFRACTION81.71
3.98-4.120.34522010.26713735X-RAY DIFFRACTION91.51
4.12-4.290.33932370.25263848X-RAY DIFFRACTION95.07
4.29-4.480.27092150.24913952X-RAY DIFFRACTION96.57
4.48-4.720.29752170.24124058X-RAY DIFFRACTION98.41
4.72-5.020.28921940.23474044X-RAY DIFFRACTION98.95
5.02-5.40.27472040.23694097X-RAY DIFFRACTION98.99
5.4-5.950.26581870.24924118X-RAY DIFFRACTION99.1
5.95-6.80.30761960.27014125X-RAY DIFFRACTION99.38
6.8-8.570.27882230.23524135X-RAY DIFFRACTION99.27
8.57-50.170.2352120.21724226X-RAY DIFFRACTION97.35

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