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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: 8236
TitleCryo-EM structure of SpCas9-sgRNA-DNA ternary complex
Map data
SampleSpCas9-sgRNA-target DNA ternay 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
SourceStreptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / 5.2 Å resolution
AuthorsLi G / Huang Q / 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 ReportPDB-ID: 5y36

SummaryFull reportAbout validation report
DateDeposition: Jun 4, 2016 / Header (metadata) release: Aug 10, 2016 / Map release: Oct 25, 2017 / Last update: Dec 6, 2017

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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
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8236.map.gz (map file in CCP4 format, 10977 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
140 pix
1.3 Å/pix.
= 182. Å
140 pix
1.3 Å/pix.
= 182. Å
140 pix
1.3 Å/pix.
= 182. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density
Contour Level:0.0316 (by author), 0.011 (movie #1):
Minimum - Maximum-0.025383996 - 0.08724286
Average (Standard dev.)0.0006787431 (0.0060282876)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions140140140
Origin000
Limit139139139
Spacing140140140
CellA=B=C: 182 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
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
Number of components: 1
MassTheoretical: 225 kDa

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Component #1: protein, SpCas9-sgRNA-target DNA ternay complex

ProteinName: SpCas9-sgRNA-target DNA ternay complex
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
Recombinant expression: No
MassTheoretical: 225 kDa
SourceSpecies: Streptococcus pyogenes (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Vector: pet28a

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.45 mg/ml
Buffer solution: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 289 K / Humidity: 100 % / Details: blot for 4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 18000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 1300 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 592

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 57484
3D reconstructionSoftware: RELION / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 4OO8
Chain ID: 4OO8_A

Overall bvalue: 80
Output model

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