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- PDB-6ifo: Crystal structure of AcrIIA2-SpyCas9-sgRNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 6ifo
TitleCrystal structure of AcrIIA2-SpyCas9-sgRNA ternary complex
Components
  • AcrIIA2
  • CRISPR-associated endonuclease Cas9/Csn1
  • RNA (99-MER)
KeywordsRNA BINDING PROTEIN/RNA / anti-CRISPR / SpyCas9 / sgRNA / AcrIIA2 / RNA BINDING PROTEIN-RNA 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 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...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 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
Listeria monocytogenes (bacteria)
Streptococcus pyogenes M1 GAS (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.313 Å
AuthorsLiu, L. / Wang, Y.
CitationJournal: Mol. Cell / Year: 2019
Title: Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race.
Authors: Liu, L. / Yin, M. / Wang, M. / Wang, Y.
History
DepositionSep 20, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.2Nov 22, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9/Csn1
C: RNA (99-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
D: RNA (99-MER)
F: AcrIIA2
E: AcrIIA2


Theoretical massNumber of molelcules
Total (without water)409,8996
Polymers409,8996
Non-polymers00
Water00
1
A: CRISPR-associated endonuclease Cas9/Csn1
C: RNA (99-MER)
E: AcrIIA2


Theoretical massNumber of molelcules
Total (without water)204,9493
Polymers204,9493
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: CRISPR-associated endonuclease Cas9/Csn1
D: RNA (99-MER)
F: AcrIIA2


Theoretical massNumber of molelcules
Total (without water)204,9493
Polymers204,9493
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)106.155, 214.567, 131.971
Angle α, β, γ (deg.)90.00, 102.89, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 158786.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): K-12
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (99-MER)


Mass: 31994.982 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Streptococcus pyogenes M1 GAS (bacteria)
#3: Protein AcrIIA2


Mass: 14167.594 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes (bacteria) / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): K-12

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.57 Å3/Da / Density % sol: 65.59 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / pH: 6 / Details: 0.1 M Bis-Tris, pH 6.0, 15% PEG 20000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97775 Å
DetectorType: DECTRIS PILATUS 300K / Detector: PIXEL / Date: Jul 14, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97775 Å / Relative weight: 1
ReflectionResolution: 3.3→50 Å / Num. obs: 56422 / % possible obs: 98.9 % / Redundancy: 5.1 % / Rpim(I) all: 0.14 / Net I/σ(I): 5.8
Reflection shellResolution: 3.3→3.36 Å / Redundancy: 3.8 % / Mean I/σ(I) obs: 1 / Rpim(I) all: 0.623 / % possible all: 96.8

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Processing

Software
NameVersionClassification
PHENIX(1.14_3247: ???)refinement
HKL-3000data reduction
HKL-3000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4ZT0

4zt0


Resolution: 3.313→49.51 Å / SU ML: 0.41 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 25.56 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2684 2843 5.04 %
Rwork0.2224 --
obs0.2247 56422 66.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.313→49.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms23624 3808 0 0 27432
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01128295
X-RAY DIFFRACTIONf_angle_d1.51839059
X-RAY DIFFRACTIONf_dihedral_angle_d24.63711163
X-RAY DIFFRACTIONf_chiral_restr0.2244535
X-RAY DIFFRACTIONf_plane_restr0.0074357
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3126-3.3697100.65498X-RAY DIFFRACTION1
3.3697-3.431100.32867X-RAY DIFFRACTION1
3.431-3.49690.863920.259951X-RAY DIFFRACTION1
3.4969-3.56830.459170.2956171X-RAY DIFFRACTION4
3.5683-3.64590.264200.2654507X-RAY DIFFRACTION12
3.6459-3.73060.2932470.26211096X-RAY DIFFRACTION27
3.7306-3.82390.3561010.26761835X-RAY DIFFRACTION45
3.8239-3.92730.31161540.26872733X-RAY DIFFRACTION68
3.9273-4.04280.32021650.26353308X-RAY DIFFRACTION82
4.0428-4.17320.29051970.24923667X-RAY DIFFRACTION91
4.1732-4.32230.31022280.24413962X-RAY DIFFRACTION99
4.3223-4.49520.27622200.22774028X-RAY DIFFRACTION99
4.4952-4.69960.2712050.2164047X-RAY DIFFRACTION99
4.6996-4.94720.2672100.21013999X-RAY DIFFRACTION99
4.9472-5.25680.28532000.21124031X-RAY DIFFRACTION99
5.2568-5.66220.26412060.21823992X-RAY DIFFRACTION98
5.6622-6.2310.26792400.22424047X-RAY DIFFRACTION100
6.231-7.13040.27611950.22494045X-RAY DIFFRACTION99
7.1304-8.97480.222320.18914009X-RAY DIFFRACTION99
8.9748-49.51570.2092140.19164036X-RAY DIFFRACTION98

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