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- PDB-7xvq: Crystal structure of AcrIIC4 -

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Basic information

Entry
Database: PDB / ID: 7xvq
TitleCrystal structure of AcrIIC4
ComponentsUncharacterized protein
KeywordsANTIMICROBIAL PROTEIN
Function / homologyUncharacterized protein
Function and homology information
Biological speciesHaemophilus parainfluenzae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsZhang, H. / Li, X.Z. / Song, G.Y.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.
Authors: Xuzichao Li / Fumeng Liao / Jiaqi Gao / Guangyong Song / Chendi Zhang / Nan Ji / Xiaoshen Wang / Jing Wen / Jia He / Yong Wei / Heng Zhang / Zhuang Li / Guimei Yu / Hang Yin /
Abstract: CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti- ...CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.
History
DepositionMay 24, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.2Feb 26, 2025Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: Uncharacterized protein
C: Uncharacterized protein
B: Uncharacterized protein
A: Uncharacterized protein


Theoretical massNumber of molelcules
Total (without water)39,9414
Polymers39,9414
Non-polymers00
Water3,225179
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1200 Å2
ΔGint5 kcal/mol
Surface area22090 Å2
Unit cell
Length a, b, c (Å)46.917, 48.468, 49.147
Angle α, β, γ (deg.)66.800, 95.720, 65.880
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein
Uncharacterized protein


Mass: 9985.316 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haemophilus parainfluenzae (bacteria) / Gene: NCTC10672_00033, NCTC10672_02354 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A377JKY9
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 179 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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: 2.27 Å3/Da / Density % sol: 45.88 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, sitting drop / Details: Sodium malonate, Polyethylene glycol 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jul 3, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 1.8→42.94 Å / Num. obs: 28492 / % possible obs: 89.01 % / Redundancy: 1.9 % / Biso Wilson estimate: 25.71 Å2 / CC1/2: 0.994 / Net I/σ(I): 8.05
Reflection shellResolution: 1.8→1.864 Å / Num. unique obs: 2793 / CC1/2: 0.872

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Processing

Software
NameVersionClassification
PHENIX1.20_4459refinement
PHENIX1.20_4459refinement
XDSdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: alphafold2

Resolution: 1.8→42.94 Å / SU ML: 0.2767 / Cross valid method: FREE R-VALUE / σ(F): 1.98 / Phase error: 29.7576
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2848 3524 7.03 %
Rwork0.2273 46628 -
obs0.2311 28487 78.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 29.63 Å2
Refinement stepCycle: LAST / Resolution: 1.8→42.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2744 0 0 179 2923
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00742795
X-RAY DIFFRACTIONf_angle_d0.92253733
X-RAY DIFFRACTIONf_chiral_restr0.048405
X-RAY DIFFRACTIONf_plane_restr0.0068486
X-RAY DIFFRACTIONf_dihedral_angle_d4.5872353
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.820.35971190.3061505X-RAY DIFFRACTION63.99
1.82-1.850.36611300.29771830X-RAY DIFFRACTION76.92
1.85-1.880.33391580.27061919X-RAY DIFFRACTION78.08
1.88-1.90.29081370.25891838X-RAY DIFFRACTION78.12
1.9-1.940.34571210.25761850X-RAY DIFFRACTION77.48
1.94-1.970.33711380.26751861X-RAY DIFFRACTION77.24
1.97-2.010.27861300.26051831X-RAY DIFFRACTION76.66
2.01-2.040.32731500.23791809X-RAY DIFFRACTION76.34
2.04-2.090.3531590.24371774X-RAY DIFFRACTION75.69
2.09-2.130.29981480.24261798X-RAY DIFFRACTION75.08
2.13-2.180.3757960.23921610X-RAY DIFFRACTION67.32
2.18-2.230.28041570.23461872X-RAY DIFFRACTION76.97
2.23-2.30.27461490.23981927X-RAY DIFFRACTION81.28
2.3-2.360.30631330.2291939X-RAY DIFFRACTION80.06
2.36-2.440.36221420.22941868X-RAY DIFFRACTION79.38
2.44-2.530.2491400.23151934X-RAY DIFFRACTION80.26
2.53-2.630.33361500.23661916X-RAY DIFFRACTION79.83
2.63-2.750.26061460.24061849X-RAY DIFFRACTION77.15
2.75-2.890.30811170.23131809X-RAY DIFFRACTION74.48
2.89-3.070.24791380.22122045X-RAY DIFFRACTION84.74
3.07-3.310.29851550.23671943X-RAY DIFFRACTION83.32
3.31-3.640.26741600.21211957X-RAY DIFFRACTION82.57
3.64-4.170.24271300.18751814X-RAY DIFFRACTION75.35
4.17-5.250.25911600.20442114X-RAY DIFFRACTION87.73
5.25-42.940.26941610.23192016X-RAY DIFFRACTION84.64

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