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- PDB-8d2o: Structure of Acidothermus cellulolyticus Cas9 ternary complex (Po... -

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Basic information

Entry
Database: PDB / ID: 8d2o
TitleStructure of Acidothermus cellulolyticus Cas9 ternary complex (Post-cleavage 2)
Components
  • (DNA target strand (5'- ...) x 2
  • CRISPR-associated endonuclease, Csn1 family
  • DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')
  • Single guide RNA (106-MER)
KeywordsRNA BINDING PROTEIN/DNA/RNA / Cas9 / AcCas9 / Crispr / Post-cleavage 2 / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / DNA binding / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease ...CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease / HNH nucleases / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease, Csn1 family
Similarity search - Component
Biological speciesAcidothermus cellulolyticus 11B (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsRai, J. / Das, A. / Li, H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM101343 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM099604 United States
CitationJournal: Nat Catal / Year: 2023
Title: Coupled catalytic states and the role of metal coordination in Cas9.
Authors: Anuska Das / Jay Rai / Mitchell O Roth / Yuerong Shu / Megan L Medina / Mackenzie R Barakat / Hong Li /
Abstract: Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its ...Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active Cas9 complex along the reaction path at 2.2-2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of Cas9 and commonly used Cas9 in cells.
History
DepositionMay 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease, Csn1 family
B: Single guide RNA (106-MER)
X: DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')
T: DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*G)-3')
D: DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,79610
Polymers172,6755
Non-polymers1225
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA target strand (5'- ... , 2 types, 2 molecules XT

#3: DNA chain DNA target strand (5'-D(*AP*GP*CP*TP*TP*GP*GP*TP*GP*TP*AP*TP*A)-3')


Mass: 4021.632 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA target strand (5'-D(P*CP*CP*AP*GP*GP*AP*TP*CP*TP*TP*G)-3')


Mass: 3349.197 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain / DNA chain , 3 types, 3 molecules ABD

#1: Protein CRISPR-associated endonuclease, Csn1 family


Mass: 127498.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidothermus cellulolyticus 11B (bacteria)
Strain: ATCC 43068 / DSM 8971 / 11B / Gene: Acel_1951 / Production host: Escherichia coli (E. coli) / References: UniProt: A0LWB3
#2: RNA chain Single guide RNA (106-MER)


Mass: 34190.301 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidothermus cellulolyticus 11B (bacteria)
Strain: ATCC 43068 / DSM 8971 / 11B / Production host: Escherichia coli (E. coli)
#5: DNA chain DNA non-target strand (5'-D(P*AP*TP*AP*CP*AP*CP*CP*AP*AP*GP*CP*T)-3')


Mass: 3615.395 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 8 molecules

#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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-IDSource
1CryoEM Structure of AceCas9 (Post-cleavage 2)COMPLEX#1-#50MULTIPLE SOURCES
2CRISPR-associated endonuclease, Csn1 family/RNACOMPLEX#1-#31RECOMBINANT
3DNACOMPLEX#4-#51SYNTHETIC
Source (natural)Organism: Acidothermus cellulolyticus 11B (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM softwareName: RELION / Version: 4 / Category: final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169296 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 41.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00458903
ELECTRON MICROSCOPYf_angle_d0.847112655
ELECTRON MICROSCOPYf_chiral_restr0.04821477
ELECTRON MICROSCOPYf_plane_restr0.00911187
ELECTRON MICROSCOPYf_dihedral_angle_d18.10752225

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