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- PDB-8jfu: AcrIIA15 in complex with palindromic DNA substrate -

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Basic information

Entry
Database: PDB / ID: 8jfu
TitleAcrIIA15 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: 3.15 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
A: AcrIIA15
C: 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')
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)103,9358
Polymers103,9358
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)51,9684
Polymers51,9684
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)51,9684
Polymers51,9684
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)110.604, 131.388, 234.183
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 0 through 8 or (resid 9...
d_2ens_1(chain "B" and (resid 0 through 10 or (resid 11...
d_3ens_1(chain "D" and (resid 0 through 8 or (resid 9...
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_1SERSERHISHISAC0 - 1631 - 164
d_2ens_1SERSERHISHISBA0 - 1631 - 164
d_3ens_1SERSERHISHISDB0 - 1631 - 164
d_1ens_2DADADGDGEE-11 - -61 - 6
d_2ens_2DADADGDGGG-11 - -61 - 6
d_1ens_3DTDTDADAFF2 - 51 - 4
d_2ens_3DTDTDADAHH2 - 51 - 4

NCS ensembles :
ID
ens_1
ens_2
ens_3

NCS oper:
IDCodeMatrixVector
1given(0.165072645977, -0.966667023731, -0.195718897354), (-0.933708486764, -0.0892420820561, -0.346733777608), (0.31770974693, 0.239980657599, -0.91731663055)10.4663288403, 62.1910339442, 56.3135731852
2given(0.757768690676, 0.63250374661, -0.160392088196), (-0.549516298104, 0.751120450224, 0.365855036009), (0.35187845849, -0.189095425063, 0.91674667748)47.3362389672, 44.5370745507, -16.2154459557
3given(0.0863917720745, -0.921038106647, 0.379770019647), (-0.980021079072, -0.0100189175309, 0.198641148473), (-0.179151182784, -0.389343585277, -0.903502311182)38.9208861068, 1.9081414721, 75.6111066571
4given(0.0707567599958, -0.921838782895, 0.381060020555), (-0.981060597123, 0.0047437005356, 0.193642975804), (-0.180315239755, -0.38754452087, -0.904044057918)39.5168657769, 1.5682083923, 75.6705112426

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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: 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: 4.09 Å3/Da / Density % sol: 69.95 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Sodium chloride, 0.1 M HEPES-NaOH pH 7.5, 25% w/v PEG 3,350

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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: Dec 15, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97851 Å / Relative weight: 1
ReflectionResolution: 3.15→50 Å / Num. obs: 30070 / % possible obs: 99.7 % / Redundancy: 5.5 % / Biso Wilson estimate: 49.59 Å2 / CC1/2: 0.999 / Net I/σ(I): 10
Reflection shellResolution: 3.15→3.22 Å / Num. unique obs: 1942 / CC1/2: 0.604

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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: 3.15→45.13 Å / SU ML: 0.5047 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 27.7179
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2748 933 5.07 %
Rwork0.2591 17478 -
obs0.2599 18411 61.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 74.21 Å2
Refinement stepCycle: LAST / Resolution: 3.15→45.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5001 1546 0 0 6547
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00826808
X-RAY DIFFRACTIONf_angle_d1.1119542
X-RAY DIFFRACTIONf_chiral_restr0.07021062
X-RAY DIFFRACTIONf_plane_restr0.0042982
X-RAY DIFFRACTIONf_dihedral_angle_d20.7562542
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CAX-RAY DIFFRACTIONTorsion NCS1.04382790939
ens_1d_3CAX-RAY DIFFRACTIONTorsion NCS7.78820469968
ens_2d_2EEX-RAY DIFFRACTIONTorsion NCS0.665521736138
ens_3d_2FFX-RAY DIFFRACTIONTorsion NCS0.681383886099
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.15-3.310.413990.2165199X-RAY DIFFRACTION4.92
3.31-3.520.2187340.2558612X-RAY DIFFRACTION15.37
3.52-3.790.3475650.27261350X-RAY DIFFRACTION33.22
3.79-4.170.31591880.27932987X-RAY DIFFRACTION75.08
4.17-4.780.29042100.27434070X-RAY DIFFRACTION99.33
4.78-6.010.31311950.2854087X-RAY DIFFRACTION99.74
6.02-45.130.22392320.22744173X-RAY DIFFRACTION98.61

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