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

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Basic information

Entry
Database: PDB / ID: 5y36
TitleCryo-EM structure of SpCas9-sgRNA-DNA ternary complex
DescriptorCRISPR-associated endonuclease Cas9/Csn1/RNA Complex
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 / ハツカネズミ, はつかねずみ /
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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Assembly

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
Polyers215,7234
Non-polymers733
Water0
#1


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

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Components

#1: Polypeptide(L)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
References: UniProt: Q99ZW2, EC: 3.1.-.-
#2: RNA chainsingle-guide RNA


Mass: 31890.916 Da / Num. of mol.: 1
Source: (synth.) Mus musculus / mammal / ハツカネズミ, はつかねずみ /
#3: DNA chaincomplementary DNA strand


Mass: 12697.152 Da / Num. of mol.: 1
Source: (synth.) Mus musculus / mammal / ハツカネズミ, はつかねずみ /
#4: DNA chainnon-complementary DNA strand


Mass: 12546.073 Da / Num. of mol.: 1
Source: (synth.) Mus musculus / mammal / ハツカネズミ, はつかねずみ /
#5: ChemicalChemComp-MG / MAGNESIUM ION


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

Component
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
10.225MEGADALTONS1NO
20.1588MEGADALTONS1NO
332.46KILODALTONS/NANOMETER1NO
433.864KILODALTONS/NANOMETER1NO
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
41mmol/LdithiothreitolDTT1
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

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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

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Processing

EM software
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1RELION1.3PARTICLE SELECTION1
2RELION1.3IMAGE ACQUISITION1
4CTFFIND33.0CTF CORRECTION1
7NAMD2.10MODEL FITTING1
9Rosetta3.5MODEL REFINEMENT1
10RELION1.3INITIAL EULER ASSIGNMENT1
11RELION1.3FINAL EULER ASSIGNMENT1
12RELION1.3CLASSIFICATION1
13RELION1.3RECONSTRUCTION1
CTF correctionType: PHASE FLIPPING ONLY
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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