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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 / 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
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsHuang, Q. / Li, G. / Huai, C.
Funding support China, 2items
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
DepositionJul 27, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 6, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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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
Polymers215,7234
Non-polymers733
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26400 Å2
ΔGint-214 kcal/mol
Surface area98820 Å2

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Components

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


Mass: 158588.781 Da / Num. of mol.: 1 / Mutation: D10A, H840A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Plasmid: pET28a
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: RNA chain single-guide RNA


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


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


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


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1SpCas9-sgRNA-target DNA complexCOMPLEX#1-#40MULTIPLE SOURCES
2SpCas9ORGANELLE OR CELLULAR COMPONENT#11RECOMBINANTCas9 protein from Streptococcus pyogenes
3single-guide RNACOMPLEX#21MULTIPLE SOURCES99 mer RNA that guide Cas9 protein to recognize and bind to target DNA
4Target DNACOMPLEX#3-#41MULTIPLE SOURCESTarget DNA for Cas9-sgRNA binding
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.225 MDaNO
210.1588 MDaNO
3132.46 kDa/nmNO
4133.864 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Streptococcus pyogenes serotype M1 (bacteria)301447
32Streptococcus pyogenes serotype M1 (bacteria)301447
43Streptococcus pyogenes serotype M1 (bacteria)301447
54Streptococcus pyogenes serotype M1 (bacteria)301447
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
32Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
43Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
54Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.5
Details: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
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 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: dCas9 protein and sgRNA, DNA were cultured at 37 degree centigrade to form a complex, and then monodisperased at 18 degree centigrade overnight.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K / Details: blot for 4 seconds before pluging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 X / 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) / Num. 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
4CTFFIND33CTF correction
7NAMD2.1model fitting
9Rosetta3.5model refinement
10RELION1.3initial Euler assignment
11RELION1.3final Euler assignment
12RELION1.3classification
13RELION1.33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 66845
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57484 / Symmetry type: POINT
Atomic model buildingB value: 80 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 4OO8

4oo8


Pdb chain-ID: A / Accession code: 4OO8 / Pdb chain residue range: 1-1368 / Source name: PDB / Type: experimental model

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