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- PDB-5y36: Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex -

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Database: PDB / ID: 5y36
TitleCryo-EM structure of SpCas9-sgRNA-DNA ternary complex
  • CRISPR-associated endonuclease Cas9/Csn1
  • complementary DNA strand
  • non-complementary DNA strand
  • single-guide RNA
KeywordsHYDROLASE/RNA/DNA / Genome editting / CRIPSR-Cas9 / DNA cleavage mechanism / HYDROLASE-DNA-RNA complex / HYDROLASE-RNA-DNA complex
Specimen sourceStreptococcus pyogenes serotype m1 / bacteria
Mus musculus / mammal / House mouse (Wikipedia) /
MethodElectron microscopy (5.2 Å resolution / Particle / Single particle)
AuthorsHuang, Q. / Li, G. / Huai, C.
CitationNat Commun, 2017, 8, 1375-1375

Nat Commun, 2017, 8, 1375-1375 Yorodumi Papers
Structural insights into DNA cleavage activation of CRISPR-Cas9 system.
Cong Huai / Gan Li / Ruijie Yao / Yingyi Zhang / Mi Cao / Liangliang Kong / Chenqiang Jia / Hui Yuan / Hongyan Chen / Daru Lu / Qiang Huang

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 27, 2017 / Release: Dec 6, 2017

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Deposited unit
A: CRISPR-associated endonuclease Cas9/Csn1
B: single-guide RNA
C: complementary DNA strand
D: non-complementary DNA strand
hetero molecules

Theoretical massNumber of molelcules
Total (without water)215,7967

TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)26400
ΔGint (kcal/M)-214
Surface area (Å2)98820


#1: Protein/peptide CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9

Mass: 158588.781 Da / Num. of mol.: 1 / Mutation: D10A, H840A
Source: (gene. exp.) Streptococcus pyogenes serotype m1 / bacteria
Gene: cas9, csn1, SPy_1046 / Plasmid name: pET28a / Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG'
References: UniProt: Q99ZW2, EC: 3.1.-.- (Acting on Ester Bonds)
#2: RNA chain single-guide RNA

Mass: 31890.916 Da / Num. of mol.: 1
Source: (synth.) Mus musculus / mammal / House mouse (Wikipedia) /
#3: DNA chain complementary DNA strand

Mass: 12697.152 Da / Num. of mol.: 1
Source: (synth.) Mus musculus / mammal / House mouse (Wikipedia) /
#4: DNA chain non-complementary DNA strand

Mass: 12546.073 Da / Num. of mol.: 1
Source: (synth.) Mus musculus / mammal / House mouse (Wikipedia) /
#5: Chemical ChemComp-MG / MAGNESIUM ION

Mass: 24.305 Da / Num. of mol.: 3 / Formula: Mg / Wikipedia: Magnesium

Experimental details


EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

Sample preparation

IDNameTypeEntity IDParent IDSourceDetails
1SpCas9-sgRNA-target DNA complexCOMPLEX1, 2, 3, 40MULTIPLE SOURCES
2SpCas9ORGANELLE OR CELLULAR COMPONENT11RECOMBINANTCas9 protein from Streptococcus pyogenes
3single-guide RNACOMPLEX21MULTIPLE SOURCES99 mer RNA that guide Cas9 protein to recognize and bind to target DNA
4Target DNACOMPLEX3, 41MULTIPLE SOURCESTarget DNA for Cas9-sgRNA binding
Molecular weight
IDValueUnitsEntity assembly IDExperimental value
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
21301447Streptococcus pyogenes serotype M1
32301447Streptococcus pyogenes serotype M1
43301447Streptococcus pyogenes serotype M1
54301447Streptococcus pyogenes serotype M1
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
21866768Escherichia coli 'BL21-Gold(DE3)pLysS AG'
32866768Escherichia coli 'BL21-Gold(DE3)pLysS AG'
43866768Escherichia coli 'BL21-Gold(DE3)pLysS AG'
54866768Escherichia coli 'BL21-Gold(DE3)pLysS AG'
Buffer solutionDetails: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
pH: 7.5
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mmol/Ltrihydroxymethyl aminomethaneTris1
2100mmol/Lpotassium chlorideKCl1
35mmol/Lmagnesium chlorideMgCl21
SpecimenConc.: 0.45 mg/ml
Details: dCas9 protein and sgRNA, DNA were cultured at 37 degree centigrade to form a complex, and then monodisperased at 18 degree centigrade overnight.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 kelvins / Details: blot for 4 seconds before pluging

Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 18000 / Nominal defocus min: 1300 nm / Calibrated defocus max: 3500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 10 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of real images: 592
Image scansDimension width: 3710 / Dimension height: 3838 / Movie frames/image: 38


EM software
Particle selectionNumber of particles selected: 66845
SymmetryPoint symmetry: C1
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 57484 / Symmetry type: POINT
Atomic model buildingOverall b value: 80 / Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 4OO8
Pdb chain ID: A / Pdb chain residue range: 1-1368

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