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- PDB-8kah: Crystal structure of SpyCas9-crRNA-tracrRNA complex bound to 18nt... -

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Basic information

Entry
Database: PDB / ID: 8kah
TitleCrystal structure of SpyCas9-crRNA-tracrRNA complex bound to 18nt target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (26-MER)
  • DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
  • RNA (34-MER)
  • RNA (65-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, 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
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: 3.36 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: 2025
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
Revision 1.2Apr 23, 2025Group: Database references / Structure summary / Category: citation / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (34-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (26-MER)
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
E: RNA (34-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (26-MER)
H: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
I: RNA (65-MER)
J: RNA (65-MER)


Theoretical massNumber of molelcules
Total (without water)403,47010
Polymers403,47010
Non-polymers00
Water00
1
A: RNA (34-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (26-MER)
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
I: RNA (65-MER)


Theoretical massNumber of molelcules
Total (without water)201,7355
Polymers201,7355
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19760 Å2
ΔGint-159 kcal/mol
Surface area81190 Å2
MethodPISA
2
E: RNA (34-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (26-MER)
H: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
J: RNA (65-MER)


Theoretical massNumber of molelcules
Total (without water)201,7355
Polymers201,7355
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19030 Å2
ΔGint-163 kcal/mol
Surface area81840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)145.214, 130.605, 148.734
Angle α, β, γ (deg.)90.000, 104.075, 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
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
12
22
13
23
14
24
15
25

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
111AAGG(chain 'A' and resid 3 through 34)AA3 - 343 - 34
221AAGGchain 'E'EE3 - 343 - 34
132LYSLYSSERSER(chain 'B' and (resid 4 through 99 or (resid 100...BB4 - 2044 - 204
142ASPASPSERSER(chain 'B' and (resid 4 through 99 or (resid 100...BB207 - 213207 - 213
152ARGARGASPASP(chain 'B' and (resid 4 through 99 or (resid 100...BB220 - 261220 - 261
162SERSERSERSER(chain 'B' and (resid 4 through 99 or (resid 100...BB267 - 714267 - 714
172GLYGLYARGARG(chain 'B' and (resid 4 through 99 or (resid 100...BB717 - 765717 - 765
182ASNASNGLNGLN(chain 'B' and (resid 4 through 99 or (resid 100...BB776 - 844776 - 844
192ASPASPASPASP(chain 'B' and (resid 4 through 99 or (resid 100...BB849 - 1012849 - 1012
1102LYSLYSTYRTYR(chain 'B' and (resid 4 through 99 or (resid 100...BB1014 - 10161014 - 1016
1112VALVALLYSLYS(chain 'B' and (resid 4 through 99 or (resid 100...BB1018 - 10201018 - 1020
1122PHEPHELEULEU(chain 'B' and (resid 4 through 99 or (resid 100...BB1038 - 10521038 - 1052
1132LYSLYSGLNGLN(chain 'B' and (resid 4 through 99 or (resid 100...BB1059 - 13641059 - 1364
2142LYSLYSSERSER(chain 'F' and (resid 4 through 305 or (resid 306...FF4 - 2044 - 204
2152ASPASPSERSER(chain 'F' and (resid 4 through 305 or (resid 306...FF207 - 213207 - 213
2162ARGARGASPASP(chain 'F' and (resid 4 through 305 or (resid 306...FF220 - 261220 - 261
2172SERSERSERSER(chain 'F' and (resid 4 through 305 or (resid 306...FF267 - 714267 - 714
2182GLYGLYARGARG(chain 'F' and (resid 4 through 305 or (resid 306...FF717 - 765717 - 765
2192ASNASNGLNGLN(chain 'F' and (resid 4 through 305 or (resid 306...FF776 - 844776 - 844
2202ASPASPVALVAL(chain 'F' and (resid 4 through 305 or (resid 306...FF849 - 1015849 - 1015
2212VALVALLYSLYS(chain 'F' and (resid 4 through 305 or (resid 306...FF1018 - 10201018 - 1020
2222PHEPHELEULEU(chain 'F' and (resid 4 through 305 or (resid 306...FF1038 - 10521038 - 1052
2232LYSLYSGLNGLN(chain 'F' and (resid 4 through 305 or (resid 306...FF1059 - 13641059 - 1364
1243DCDCDTDTchain 'C'CC1 - 261 - 26
2253DCDCDTDTchain 'G'GG1 - 261 - 26
1264DTDTDGDGchain 'D'DD2 - 121 - 11
2274DTDTDGDGchain 'H'HH2 - 121 - 11
1285GGUUchain 'I'II36 - 982 - 64
2295GGUUchain 'J'JJ36 - 982 - 64

NCS ensembles :
ID
1
2
3
4
5

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Components

#1: RNA chain RNA (34-MER)


Mass: 10912.471 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: crRNA
Source: (synth.) Streptococcus pyogenes serotype M1 (bacteria)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158588.781 Da / Num. of mol.: 2 / Mutation: A10D,A840H
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
#3: DNA chain DNA (26-MER)


Mass: 7871.114 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)
#5: RNA chain RNA (65-MER)


Mass: 20968.490 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: tracrRNA
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Production host: Escherichia coli DH5[alpha] (bacteria)
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: 3.39 Å3/Da / Density % sol: 63.73 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 1.0 M Sodium chloride, 0.1 M Sodium citrate pH 6.0, 13.25% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.97853 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 24, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 3.28→50 Å / Num. obs: 89919 / % possible obs: 99.6 % / Redundancy: 5.1 % / Biso Wilson estimate: 62.32 Å2 / Rpim(I) all: 0.096 / Net I/σ(I): 7
Reflection shellResolution: 3.28→3.34 Å / Redundancy: 4.4 % / Num. unique obs: 4072 / Rpim(I) all: 0.457 / % possible all: 99.3

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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 / Resolution: 3.36→48.23 Å / SU ML: 0.3706 / Cross valid method: FREE R-VALUE / σ(F): 1.4 / Phase error: 23.6586
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2547 3090 3.44 %
Rwork0.2298 86829 -
obs0.2307 89919 59.41 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 81.95 Å2
Refinement stepCycle: LAST / Resolution: 3.36→48.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21427 5598 0 0 27025
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01228054
X-RAY DIFFRACTIONf_angle_d1.560639023
X-RAY DIFFRACTIONf_chiral_restr0.1094509
X-RAY DIFFRACTIONf_plane_restr0.00934027
X-RAY DIFFRACTIONf_dihedral_angle_d19.946111368
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.36-3.410.938800.385819X-RAY DIFFRACTION0.28
3.41-3.470.635830.260940X-RAY DIFFRACTION0.65
3.47-3.530.299680.2912249X-RAY DIFFRACTION3.8
3.53-3.590.311170.2633450X-RAY DIFFRACTION6.82
3.59-3.660.3285280.2813845X-RAY DIFFRACTION12.71
3.66-3.730.3099580.28461574X-RAY DIFFRACTION23.49
3.73-3.810.3158850.28692458X-RAY DIFFRACTION37.35
3.81-3.90.33121090.29233088X-RAY DIFFRACTION46.45
3.9-40.29541110.28373330X-RAY DIFFRACTION50.15
4-4.110.29971270.26233665X-RAY DIFFRACTION55.01
4.11-4.230.25621550.24424022X-RAY DIFFRACTION60.62
4.23-4.370.23411550.23324560X-RAY DIFFRACTION68.02
4.37-4.520.26491720.22815104X-RAY DIFFRACTION76.89
4.52-4.70.26451880.22015592X-RAY DIFFRACTION84.7
4.7-4.920.26742240.22166128X-RAY DIFFRACTION92.42
4.92-5.180.24732340.22296414X-RAY DIFFRACTION96.07
5.18-5.50.24892340.22326508X-RAY DIFFRACTION97.99
5.5-5.920.24152430.22676565X-RAY DIFFRACTION98.87
5.92-6.520.22152350.24336574X-RAY DIFFRACTION99.4
6.52-7.460.26522220.23576625X-RAY DIFFRACTION99.43
7.46-9.390.19032420.20396604X-RAY DIFFRACTION99.46
9.39-48.230.2662400.20166415X-RAY DIFFRACTION96.72

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