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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
Components
  • 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
Function / homologyCRISPR-associated endonuclease Cas9, PAM-interacting domain / REC lobe of CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain profile. / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / HNH nuclease / CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / Cas9-type HNH domain ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / REC lobe of CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain profile. / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / HNH nuclease / CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / Cas9-type HNH domain / HNH endonuclease / maintenance of CRISPR repeat elements / endodeoxyribonuclease activity / 3'-5' exonuclease activity / defense response to virus / Acting on Ester Bonds / RNA binding / DNA binding / metal ion binding / CRISPR-associated endonuclease Cas9/Csn1
Function and homology information
Specimen sourceStreptococcus pyogenes serotype M1 (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 5.2 Å resolution
AuthorsHuang, Q. / Li, G. / Huai, C.
CitationJournal: Nat Commun / Year: 2017
Title: Structural insights into DNA cleavage activation of CRISPR-Cas9 system.
Authors: 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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Structure visualization

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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: 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' (bacteria)
References: UniProt: Q99ZW2, Acting on Ester Bonds
#2: RNA chain single-guide RNA


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


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


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


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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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
IDValueEntity assembly IDExperimental value
10.225 MDa1NO
20.1588 MDa1NO
332.46 kDa/nm1NO
433.864 kDa/nm1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
21301447Streptococcus pyogenes serotype M1 (bacteria)
32301447Streptococcus pyogenes serotype M1 (bacteria)
43301447Streptococcus pyogenes serotype M1 (bacteria)
54301447Streptococcus pyogenes serotype M1 (bacteria)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
21866768Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
32866768Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
43866768Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
54866768Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionDetails: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
pH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
120 mmol/Ltrihydroxymethyl aminomethaneTris1
2100 mmol/Lpotassium chlorideKCl1
35 mmol/Lmagnesium chlorideMgCl21
41 mmol/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 FIELDBright-field microscopy / 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 scansWidth: 3710 / Height: 3838 / Movie frames/image: 38

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Processing

EM software
IDNameVersionCategory
1RELION1.3particle selection
2RELION1.3image acquisition
4CTFFIND33.0CTF correction
7NAMD2.10model fitting
9Rosetta3.5model refinement
10RELION1.3initial Euler assignment
11RELION1.3final Euler assignment
12RELION1.3classification
13RELION1.33D reconstruction
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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