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- PDB-5vw1: Crystal structure of SpyCas9-sgRNA-AcrIIA4 ternary complex -

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Basic information

Entry
Database: PDB / ID: 5vw1
TitleCrystal structure of SpyCas9-sgRNA-AcrIIA4 ternary complex
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • anti-CRISPR protein AcrIIA4
  • sgRNASubgenomic mRNA
KeywordsHYDROLASE / Type II CRISPR-Cas endonculease: Cas9: Structure: Endonuclease: anti-CRISPR protein: Inhibition of Cas9: RuvC catalytic pocket: Sequence-specific PAM recognition: Genome editing tool
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
DI(HYDROXYETHYL)ETHER / RNA / RNA (> 10) / Uncharacterized protein / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
Listeria monocytogenes serotype 4a
Listeria monocytogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.598 Å
AuthorsYang, H. / Patel, D.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM104962 United States
Memorial Sloan Kettering Cancer Center Core GrantP30CA008748 United States
CitationJournal: Mol. Cell / Year: 2017
Title: Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.
Authors: Yang, H. / Patel, D.J.
History
DepositionMay 21, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 28, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Oct 4, 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: sgRNA
B: anti-CRISPR protein AcrIIA4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,1469
Polymers196,4593
Non-polymers6876
Water3,423190
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16090 Å2
ΔGint-88 kcal/mol
Surface area73910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)72.194, 101.242, 303.563
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 158732.891 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: pRSFDuet-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#3: Protein anti-CRISPR protein AcrIIA4 /


Mass: 10326.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes serotype 4a (strain M7) (bacteria)
Strain: M7 / Gene: LMM7_0114 / Plasmid: pRSF-Duet-SUMO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: A0A0E0UT28

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RNA chain , 1 types, 1 molecules C

#2: RNA chain sgRNA / Subgenomic mRNA


Mass: 27400.332 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: sgRNA was prepared by in vitro transcription / Source: (synth.) Listeria monocytogenes (bacteria)

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Non-polymers , 5 types, 196 molecules

#4: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#5: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL / Polyethylene glycol


Mass: 194.226 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#6: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 190 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailshypothetical protein LMOG_02993 [Listeria monocytogenes J0161] GenBank: AEO04689.1

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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: 2.84 Å3/Da / Density % sol: 56.66 % / Mosaicity: 0.305 °
Crystal growTemperature: 293 K / Method: evaporation / pH: 7.2 / Details: 0.1 M HEPES (pH 7.5), and 15% PEG3350 (v/v)

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Mar 23, 2016 / Details: LR-Design detector positioner
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.598→50 Å / Num. obs: 65656 / % possible obs: 94.6 % / Redundancy: 4.3 % / Biso Wilson estimate: 48.71 Å2 / Rmerge(I) obs: 0.092 / Rpim(I) all: 0.048 / Rrim(I) all: 0.104 / Χ2: 0.875 / Net I/σ(I): 7.1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.6-2.693.40.4970.8050.2760.5740.60372.1
2.69-2.83.70.4280.8440.2340.4910.61685.8
2.8-2.933.90.380.8890.2090.4370.62395.6
2.93-3.084.20.3240.9290.1740.370.65898.9
3.08-3.283.90.2260.9620.1250.260.72598.3
3.28-3.534.70.1450.9870.0730.1630.82199.7
3.53-3.884.80.10.9930.050.1120.95999.4
3.88-4.454.60.0690.9960.0350.0781.0298.9
4.45-5.64.60.0550.9970.0280.0621.07298.5
5.6-504.80.0420.9990.0210.0481.25997.8

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
HKL-2000data scaling
PDB_EXTRACT3.22data extraction
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4ZT0

4zt0


Resolution: 2.598→48.021 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 25.17 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2261 3300 5.03 %
Rwork0.1781 --
obs0.1805 65587 94.14 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.598→48.021 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11613 1562 45 190 13410
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00913603
X-RAY DIFFRACTIONf_angle_d1.05318684
X-RAY DIFFRACTIONf_dihedral_angle_d23.4635355
X-RAY DIFFRACTIONf_chiral_restr0.0642146
X-RAY DIFFRACTIONf_plane_restr0.0052128
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5979-2.6350.3474940.28291514X-RAY DIFFRACTION57
2.635-2.67440.3333980.27122004X-RAY DIFFRACTION73
2.6744-2.71620.33611010.25552184X-RAY DIFFRACTION81
2.7162-2.76070.31461160.25932359X-RAY DIFFRACTION85
2.7607-2.80830.29141220.24882461X-RAY DIFFRACTION91
2.8083-2.85930.33591340.2452557X-RAY DIFFRACTION94
2.8593-2.91430.32651520.24982618X-RAY DIFFRACTION97
2.9143-2.97380.30531440.2362714X-RAY DIFFRACTION99
2.9738-3.03850.25921250.2332717X-RAY DIFFRACTION99
3.0385-3.10910.30491440.22412690X-RAY DIFFRACTION99
3.1091-3.18690.26361430.21942707X-RAY DIFFRACTION99
3.1869-3.2730.25221470.21622678X-RAY DIFFRACTION98
3.273-3.36930.23441440.20792714X-RAY DIFFRACTION100
3.3693-3.4780.26911360.19442739X-RAY DIFFRACTION100
3.478-3.60230.24641490.18242743X-RAY DIFFRACTION100
3.6023-3.74650.2581400.16722733X-RAY DIFFRACTION100
3.7465-3.91690.19121470.15882726X-RAY DIFFRACTION99
3.9169-4.12330.20641620.16012715X-RAY DIFFRACTION99
4.1233-4.38150.19971340.14342750X-RAY DIFFRACTION99
4.3815-4.71950.18191460.13822729X-RAY DIFFRACTION98
4.7195-5.19390.18851560.13722753X-RAY DIFFRACTION98
5.1939-5.94430.21121510.15842797X-RAY DIFFRACTION100
5.9443-7.48470.20121520.1692800X-RAY DIFFRACTION99
7.4847-48.02890.15941630.14042885X-RAY DIFFRACTION96
Refinement TLS params.Method: refined / Origin x: -22.2824 Å / Origin y: -27.0876 Å / Origin z: 42.5557 Å
111213212223313233
T0.4521 Å2-0.0135 Å20.0253 Å2-0.2385 Å20.0128 Å2--0.284 Å2
L0.4085 °2-0.1538 °20.1809 °2-0.9566 °20.1396 °2--0.4267 °2
S-0.0017 Å °-0.0649 Å °0.0053 Å °-0.0025 Å °-0.0265 Å °0.0271 Å °-0.0167 Å °-0.0668 Å °0.028 Å °
Refinement TLS groupSelection details: all

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