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- EMDB-8236: Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-8236
TitleCryo-EM structure of SpCas9-sgRNA-DNA ternary complex
Map dataNone
Sample
  • Complex: SpCas9-sgRNA-target DNA ternay 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
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsLi G / Huang Q / Huai C
Funding support China, 2 items
OrganizationGrant numberCountry
The Natural Science Foundation of China91430112 China
The Shanghai Natural Science Foundation13ZR1402400 China
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 /
Abstract: CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease ...CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.
History
DepositionJun 4, 2016-
Header (metadata) releaseAug 10, 2016-
Map releaseOct 25, 2017-
UpdateJan 22, 2020-
Current statusJan 22, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5y36
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8236.png

Downloads & links

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Map

FileDownload / File: emd_8236.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 0.0316 / Movie #1: 0.011
Minimum - Maximum-0.025383996 - 0.08724286
Average (Standard dev.)0.0006787431 (±0.0060282876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 182.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z182.000182.000182.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0250.0870.001

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Supplemental data

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Sample components

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Entire : SpCas9-sgRNA-target DNA ternay complex

EntireName: SpCas9-sgRNA-target DNA ternay complex
Components
  • Complex: SpCas9-sgRNA-target DNA ternay complex

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Supramolecule #1: SpCas9-sgRNA-target DNA ternay complex

SupramoleculeName: SpCas9-sgRNA-target DNA ternay complex / type: complex / ID: 1 / Parent: 0
Details: S. pyogenes Cas9(D10A, H840A) in complex with a 55-mer target DNA from the fumarylacetoacetate hydrolase (FAH) gene and the corresponding 98-nucleotide sgRNA
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 225 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.45 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTristrihydroxymethyl aminomethane
100.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
1.0 mMDTTdithiothreitol

Details: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.
DetailsSpCas9 protein, sgRNA, and target DNA were cultured at 37 degree centigrade to form a complex, and then monodisperased at 18 degree centigrade overnight.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 18000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number real images: 592 / Average electron dose: 10.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 66845
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4un3


Details: The structure was low-pass filtered to 60 Angstrom.
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 57484
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4oo8


Chain - Chain ID: A / Chain - Residue range: 3-1366
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 80 / Target criteria: Correlation coefficient
Output model

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

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