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- PDB-8jfo: Crystal structure of anti-CRISPR protein AcrIIA15 -

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Basic information

Entry
Database: PDB / ID: 8jfo
TitleCrystal structure of anti-CRISPR protein AcrIIA15
ComponentsAcrIIA15
KeywordsVIRAL PROTEIN / IIA type anti-crispr protein
Biological speciesStaphylococcus delphini (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsDeng, X. / Wang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31930065 China
National Natural Science Foundation of China (NSFC)31725008 China
CitationJournal: Nat Commun / Year: 2024
Title: An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding.
Authors: Xieshuting Deng / Wei Sun / Xueyan Li / Jiuyu Wang / Zhi Cheng / Gang Sheng / Yanli Wang /
Abstract: AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two ...AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two activities remains unclear. Here, we determined the cryo-EM structure of AcrIIA15 in complex with SaCas9-sgRNA to reveal the inhibitory mechanism of the Acr's C-terminal domain (CTD) in mimicking dsDNA to block protospacer adjacent motif (PAM) recognition. For the N-terminal domain (NTD), our crystal structures of the AcrIIA15-promoter DNA show that AcrIIA15 dimerizes through its NTD to recognize double-stranded (ds) DNA. Further, AcrIIA15 can simultaneously bind to both SaCas9-sgRNA and promoter DNA, creating a supercomplex of two Cas9s bound to two CTDs converging on a dimer of the NTD bound to a dsDNA. These findings shed light on AcrIIA15's inhibitory mechanisms and its autoregulation of transcription, enhancing our understanding of phage-host interactions and CRISPR defense.
History
DepositionMay 18, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / pdbx_initial_refinement_model / refine
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _refine.pdbx_method_to_determine_struct / _refine.pdbx_starting_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: AcrIIA15
D: AcrIIA15
A: AcrIIA15
C: AcrIIA15


Theoretical massNumber of molelcules
Total (without water)80,6444
Polymers80,6444
Non-polymers00
Water7,494416
1
B: AcrIIA15
D: AcrIIA15


Theoretical massNumber of molelcules
Total (without water)40,3222
Polymers40,3222
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: AcrIIA15
C: AcrIIA15


Theoretical massNumber of molelcules
Total (without water)40,3222
Polymers40,3222
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)88.194, 88.194, 145.649
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number76
Space group name H-MP41
Space group name HallP4w
Symmetry operation#1: x,y,z
#2: -y,x,z+1/4
#3: y,-x,z+3/4
#4: -x,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 0 through 15 or (resid 16...
d_2ens_1(chain "B" and (resid 0 through 20 or (resid 21...
d_3ens_1(chain "C" and ((resid 0 and (name N or name...
d_4ens_1(chain "D" and (resid 0 through 2 or (resid 3...

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: SER / Beg label comp-ID: SER / End auth comp-ID: GLU / End label comp-ID: GLU / Auth seq-ID: 0 - 170 / Label seq-ID: 1 - 171

Dom-IDAuth asym-IDLabel asym-ID
d_1AC
d_2BA
d_3CD
d_4DB

NCS oper:
IDCodeMatrixVector
1given(-0.000637258641962, -0.99999936376, -0.000930796431865), (-0.99999930426, 0.000636334083678, 0.000993256684069), (-0.000992663754624, 0.000931428745678, -0.999999073529)44.1323821545, 44.1148598827, 0.345218609104
2given(-0.339491622754, 0.655212970719, 0.674863987098), (0.873788251694, -0.0459105892902, 0.484134598013), (0.34819467153, 0.754047863728, -0.556931135711)58.8643021548, -31.5601296699, -52.5297639159
3given(-0.873424551873, 0.0460564295391, -0.48477660575), (0.339969408558, -0.655072207251, -0.674760108877), (-0.348640722563, -0.754161261658, 0.556498371952)75.6374029943, -14.7941327371, 52.8621012127

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Components

#1: Protein
AcrIIA15


Mass: 20160.963 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus delphini (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 416 / Source method: isolated from a natural source / Formula: H2O

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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: 3.51 Å3/Da / Density % sol: 64.98 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Trimethylamine N-oxide dihydrate, 0.1 M Tris pH 8.82, 18% w/v PEG 2,000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL02U1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 S 9M / Detector: PIXEL / Date: Aug 28, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.3→34.68 Å / Num. obs: 49185 / % possible obs: 99.7 % / Redundancy: 5.8 % / Biso Wilson estimate: 31.01 Å2 / CC1/2: 0.996 / Net I/σ(I): 21.6
Reflection shellResolution: 2.3→2.34 Å / Num. unique obs: 2431 / CC1/2: 0.645

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: previously solved AcrIIA15(CTD) structure using SAD

Resolution: 2.3→34.68 Å / SU ML: 0.2826 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.3436
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2327 2334 5.06 %
Rwork0.222 43774 -
obs0.2225 46108 93.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 43.59 Å2
Refinement stepCycle: LAST / Resolution: 2.3→34.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5506 0 0 416 5922
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00995595
X-RAY DIFFRACTIONf_angle_d1.30667560
X-RAY DIFFRACTIONf_chiral_restr0.0829815
X-RAY DIFFRACTIONf_plane_restr0.0049995
X-RAY DIFFRACTIONf_dihedral_angle_d13.4662048
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CAX-RAY DIFFRACTIONTorsion NCS0.439957777042
ens_1d_3CAX-RAY DIFFRACTIONTorsion NCS7.9020101094
ens_1d_4CAX-RAY DIFFRACTIONTorsion NCS7.90352081311
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.350.3313800.28921489X-RAY DIFFRACTION53.9
2.35-2.40.34521040.28571943X-RAY DIFFRACTION70.3
2.4-2.450.3793880.28632324X-RAY DIFFRACTION84.37
2.45-2.520.30191430.28322540X-RAY DIFFRACTION92.29
2.52-2.580.30171400.26332624X-RAY DIFFRACTION95.77
2.58-2.660.25491490.25142684X-RAY DIFFRACTION98.3
2.66-2.750.25661290.26812718X-RAY DIFFRACTION98.34
2.75-2.840.30441220.26582752X-RAY DIFFRACTION99.1
2.84-2.960.27581810.26332702X-RAY DIFFRACTION99.62
2.96-3.090.27061460.25572709X-RAY DIFFRACTION99.62
3.09-3.250.2421320.23262753X-RAY DIFFRACTION99.65
3.26-3.460.23661470.21252756X-RAY DIFFRACTION99.97
3.46-3.730.22121590.21122728X-RAY DIFFRACTION100
3.73-4.10.18631520.19612753X-RAY DIFFRACTION99.93
4.1-4.690.1891530.17562765X-RAY DIFFRACTION99.93
4.69-5.910.21631610.19812744X-RAY DIFFRACTION99.93
5.91-34.680.18411480.19272790X-RAY DIFFRACTION99.63

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