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- PDB-8jfr: N-terminal domain of AcrIIA15 in complex with palindromic DNA sub... -

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Basic information

Entry
Database: PDB / ID: 8jfr
TitleN-terminal domain of AcrIIA15 in complex with palindromic DNA substrate
Components
  • AcrIIA15
  • DNA (5'-D(*AP*TP*TP*AP*TP*GP*AP*CP*AP*AP*AP*TP*GP*TP*CP*AP*TP*AP*G)-3')
  • DNA (5'-D(*TP*CP*TP*AP*TP*GP*AP*CP*AP*TP*TP*TP*GP*TP*CP*AP*TP*AP*A)-3')
KeywordsVIRAL PROTEIN / IIA type anti-crispr protein
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesStaphylococcus delphini (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 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

Downloads & links

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Assembly

Deposited unit
B: AcrIIA15
D: AcrIIA15
E: DNA (5'-D(*AP*TP*TP*AP*TP*GP*AP*CP*AP*AP*AP*TP*GP*TP*CP*AP*TP*AP*G)-3')
F: DNA (5'-D(*TP*CP*TP*AP*TP*GP*AP*CP*AP*TP*TP*TP*GP*TP*CP*AP*TP*AP*A)-3')
A: AcrIIA15
C: AcrIIA15
G: DNA (5'-D(*AP*TP*TP*AP*TP*GP*AP*CP*AP*AP*AP*TP*GP*TP*CP*AP*TP*AP*G)-3')
H: DNA (5'-D(*TP*CP*TP*AP*TP*GP*AP*CP*AP*TP*TP*TP*GP*TP*CP*AP*TP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)51,6768
Polymers51,6768
Non-polymers00
Water00
1
B: AcrIIA15
D: AcrIIA15
E: DNA (5'-D(*AP*TP*TP*AP*TP*GP*AP*CP*AP*AP*AP*TP*GP*TP*CP*AP*TP*AP*G)-3')
F: DNA (5'-D(*TP*CP*TP*AP*TP*GP*AP*CP*AP*TP*TP*TP*GP*TP*CP*AP*TP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)25,8384
Polymers25,8384
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: AcrIIA15
C: AcrIIA15
G: DNA (5'-D(*AP*TP*TP*AP*TP*GP*AP*CP*AP*AP*AP*TP*GP*TP*CP*AP*TP*AP*G)-3')
H: DNA (5'-D(*TP*CP*TP*AP*TP*GP*AP*CP*AP*TP*TP*TP*GP*TP*CP*AP*TP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)25,8384
Polymers25,8384
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)58.712, 58.712, 240.593
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number78
Space group name H-MP43
Space group name HallP4cw
Symmetry operation#1: x,y,z
#2: -y,x,z+3/4
#3: y,-x,z+1/4
#4: -x,-y,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 0 through 2 or (resid 3...
d_2ens_1(chain "B" and (resid 0 through 2 or (resid 3...
d_3ens_1(chain "C" and (resid 0 through 20 or (resid 21...
d_4ens_1(chain "D" and (resid 0 through 20 or (resid 21...
d_1ens_2chain "E"
d_2ens_2chain "G"
d_1ens_3chain "F"
d_2ens_3chain "H"

NCS domain segments:

Component-ID: 1

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_1ens_1SERSERVALVALAE0 - 601 - 61
d_2ens_1SERSERVALVALBA0 - 601 - 61
d_3ens_1SERSERVALVALCF0 - 601 - 61
d_4ens_1SERSERVALVALDB0 - 601 - 61
d_1ens_2DADADGDGEC-11 - -61 - 6
d_2ens_2DADADGDGGG-11 - -61 - 6
d_1ens_3DTDTDADAFD2 - 51 - 4
d_2ens_3DTDTDADAHH2 - 51 - 4

NCS ensembles :
ID
ens_1
ens_2
ens_3

NCS oper:
IDCodeMatrixVector
1given(0.00174751932573, 0.999970382406, 0.00749536435513), (0.99998987855, -0.00177853078419, 0.00413275040589), (0.00414595874001, 0.00748806642997, -0.999963369273)29.5159047415, -29.2689176225, 3.63023618557
2given(-0.997899758506, 0.0634687152725, 0.012953538316), (0.0645890434651, 0.959679289089, 0.27357616409), (0.00493228522171, 0.273838244728, -0.961763114435)8.94308847984, 3.44623783503, -27.1948592083
3given(0.0714461994628, 0.958152121379, 0.277200203608), (-0.997439151315, 0.0695376271543, 0.0167229732269), (-0.00325269213243, -0.277685128711, 0.960666637958)32.8321359606, -20.2404982255, 30.7762194349
4given(-0.00645032810186, 0.999977750835, 0.00170032397677), (0.999978852299, 0.00644890592347, 0.000840576012772), (0.000829592081292, 0.00170571000991, -0.999998201164)29.5244721234, -29.2342178972, 3.60351392991
5given(-0.00391779223274, 0.999989471886, 0.00238893707569), (0.999991585541, 0.0039148720861, 0.00122581577437), (0.00121645048577, 0.0023937194656, -0.999996395171)29.4553044703, -29.2982186364, 3.58168903069

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Components

#1: Protein
AcrIIA15


Mass: 7096.128 Da / Num. of mol.: 4 / Fragment: N-terminal domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus delphini (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: DNA chain DNA (5'-D(*AP*TP*TP*AP*TP*GP*AP*CP*AP*AP*AP*TP*GP*TP*CP*AP*TP*AP*G)-3')


Mass: 5851.835 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Staphylococcus delphini (bacteria)
#3: DNA chain DNA (5'-D(*TP*CP*TP*AP*TP*GP*AP*CP*AP*TP*TP*TP*GP*TP*CP*AP*TP*AP*A)-3')


Mass: 5793.783 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Staphylococcus delphini (bacteria)

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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: 4.06 Å3/Da / Density % sol: 69.72 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / Details: 1.6 M sodium citrate pH 6.05

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97851 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 26, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97851 Å / Relative weight: 1
ReflectionResolution: 3.1→50 Å / Num. obs: 14903 / % possible obs: 99.9 % / Redundancy: 4.7 % / Biso Wilson estimate: 55.86 Å2 / CC1/2: 1 / Net I/σ(I): 24.9
Reflection shellResolution: 3.1→3.15 Å / Num. unique obs: 725 / CC1/2: 0.996

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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: 3.1→29.66 Å / SU ML: 0.3851 / Cross valid method: FREE R-VALUE / σ(F): 1.41 / Phase error: 29.8701
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2589 478 5.09 %
Rwork0.2462 8916 -
obs0.2468 9394 64 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 63.54 Å2
Refinement stepCycle: LAST / Resolution: 3.1→29.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1814 1546 0 0 3360
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00953550
X-RAY DIFFRACTIONf_angle_d1.29265101
X-RAY DIFFRACTIONf_chiral_restr0.0849604
X-RAY DIFFRACTIONf_plane_restr0.0046381
X-RAY DIFFRACTIONf_dihedral_angle_d25.48981425
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2EAX-RAY DIFFRACTIONTorsion NCS0.478691194526
ens_1d_3EAX-RAY DIFFRACTIONTorsion NCS0.65481654599
ens_1d_4EAX-RAY DIFFRACTIONTorsion NCS0.577109980972
ens_2d_2CEX-RAY DIFFRACTIONTorsion NCS0.535044092014
ens_3d_2DFX-RAY DIFFRACTIONTorsion NCS0.301052612688
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.550.2628710.27521374X-RAY DIFFRACTION29.8
3.55-4.470.28121610.26492900X-RAY DIFFRACTION62.52
4.47-29.660.24782460.23324642X-RAY DIFFRACTION99.35

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