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- PDB-8hns: Crystal structure of an anti-CRISPR protein AcrIIC4 in apo form -

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Entry
Database: PDB / ID: 8hns
TitleCrystal structure of an anti-CRISPR protein AcrIIC4 in apo form
Componentsanti-CRISPR protein AcrIIC4
KeywordsANTIMICROBIAL PROTEIN / anti-CRISPR protein / cleavage inhibition / dimer
Biological speciesHaemophilus parainfluenzae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.54 Å
AuthorsSun, W. / Cheng, Z. / Yang, J. / Wang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2023
Title: AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation.
Authors: Wei Sun / Zhi Cheng / Jiuyu Wang / Jing Yang / Xueyan Li / Jinlong Wang / Minxuan Chen / Xiaoqi Yang / Gang Sheng / Jizhong Lou / Yanli Wang /
Abstract: Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the ...Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.
#1: Journal: mBio / Year: 2018
Title: Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.
Authors: Lee, J. / Mir, A. / Edraki, A. / Garcia, B. / Amrani, N. / Lou, H.E. / Gainetdinov, I. / Pawluk, A. / Ibraheim, R. / Gao, X.D. / Liu, P. / Davidson, A.R. / Maxwell, K.L. / Sontheimer, E.J.
History
DepositionDec 8, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 19, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Database references / Category: citation / citation_author
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
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: anti-CRISPR protein AcrIIC4
B: anti-CRISPR protein AcrIIC4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,3294
Polymers20,1452
Non-polymers1842
Water43224
1
A: anti-CRISPR protein AcrIIC4
hetero molecules

A: anti-CRISPR protein AcrIIC4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,3294
Polymers20,1452
Non-polymers1842
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,-y,z1
Buried area4600 Å2
ΔGint-41 kcal/mol
Surface area11080 Å2
MethodPISA
2
B: anti-CRISPR protein AcrIIC4
hetero molecules

B: anti-CRISPR protein AcrIIC4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,3294
Polymers20,1452
Non-polymers1842
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,-y,z1
Buried area4670 Å2
ΔGint-37 kcal/mol
Surface area11120 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.179, 83.179, 27.814
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number75
Space group name H-MP4
Space group name HallP4
Symmetry operation#1: x,y,z
#2: -y,x,z
#3: y,-x,z
#4: -x,-y,z

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Components

#1: Protein anti-CRISPR protein AcrIIC4 /


Mass: 10072.395 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: WP_049372635.1;The first residue 'Ser' of the sample sequence is the one expressed from the vector left after tag cleavage.
Source: (gene. exp.) Haemophilus parainfluenzae (bacteria) / Gene: acrIIC4 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C3H8O3
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.41 Å3/Da / Density % sol: 48.94 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: tris, sodium chloride, sodium acetate, PEG 3350, glycerol

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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 EIGER X 16M / Detector: PIXEL / Date: Oct 11, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 2.54→50 Å / Num. obs: 6556 / % possible obs: 99.5 % / Redundancy: 7.3 % / Biso Wilson estimate: 32.47 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.042 / Net I/σ(I): 37.8
Reflection shellResolution: 2.54→2.58 Å / Rmerge(I) obs: 0.83 / Num. unique obs: 328 / CC1/2: 0.796

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
HKL-2000data reduction
HKL-2000data scaling
AutoSolphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: in-house model ( the crystal structure of a SeMet-derivative of this protein using SAD)

Resolution: 2.54→37.2 Å / SU ML: 0.2404 / Cross valid method: FREE R-VALUE / σ(F): 1.41 / Phase error: 22.6182
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2428 296 4.7 %
Rwork0.2208 6006 -
obs0.2218 6302 95.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 41.51 Å2
Refinement stepCycle: LAST / Resolution: 2.54→37.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1398 0 12 24 1434
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.02071430
X-RAY DIFFRACTIONf_angle_d1.41831910
X-RAY DIFFRACTIONf_chiral_restr0.1079208
X-RAY DIFFRACTIONf_plane_restr0.0084248
X-RAY DIFFRACTIONf_dihedral_angle_d27.8242540
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.54-3.20.28011560.28162820X-RAY DIFFRACTION92.05
3.2-37.20.22321400.19543186X-RAY DIFFRACTION99.08

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