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- PDB-8hnw: Crystal structure of HpaCas9-sgRNA surveillance complex bound to ... -

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Basic information

Entry
Database: PDB / ID: 8hnw
TitleCrystal structure of HpaCas9-sgRNA surveillance complex bound to double-stranded DNA
Components
  • CRISPR-associated endonuclease Cas9
  • Non-target strand
  • Target strand
  • sgRNA
KeywordsIMMUNE SYSTEM / Cas9 / ANTIMICROBIAL PROTEIN / dsDNA
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesHaemophilus parainfluenzae (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.41 Å
AuthorsSun, W. / Cheng, Z. / 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: Jooyoung Lee / Aamir Mir / Alireza Edraki / Bianca Garcia / Nadia Amrani / Hannah E Lou / Ildar Gainetdinov / April Pawluk / Raed Ibraheim / Xin D Gao / Pengpeng Liu / Alan R Davidson / ...Authors: Jooyoung Lee / Aamir Mir / Alireza Edraki / Bianca Garcia / Nadia Amrani / Hannah E Lou / Ildar Gainetdinov / April Pawluk / Raed Ibraheim / Xin D Gao / Pengpeng Liu / Alan R Davidson / Karen L Maxwell / Erik J Sontheimer /
Abstract: In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used ...In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of and , both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from and , show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from and reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications. As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.
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:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: sgRNA
C: Target strand
D: Non-target strand


Theoretical massNumber of molelcules
Total (without water)176,7214
Polymers176,7214
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19020 Å2
ΔGint-163 kcal/mol
Surface area57160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)158.867, 185.760, 160.880
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

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 121649.695 Da / Num. of mol.: 1 / Mutation: H581A
Source method: isolated from a genetically manipulated source
Details: One mutation H581A was introduced to inactivate the catalytic site of HNH domain of HpaCas9. 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: csn1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: F0ET08
#2: RNA chain sgRNA


Mass: 40860.125 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: RNA is originally derived from Haemophilus parainfluenzae and modified by author.
Source: (synth.) synthetic construct (others)
#3: DNA chain Target strand


Mass: 10849.030 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Haemophilus parainfluenzae (bacteria)
#4: DNA chain Non-target strand


Mass: 3362.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Haemophilus parainfluenzae (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: 4.31 Å3/Da / Density % sol: 71.44 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: tris, sodium chloride, sodium acetate, lithium sulfate, PEG 10000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.979 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jan 31, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 3.4→50 Å / Num. obs: 32947 / % possible obs: 100 % / Redundancy: 7.2 % / Biso Wilson estimate: 39.85 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.132 / Net I/σ(I): 16.64
Reflection shellResolution: 3.4→3.46 Å / Rmerge(I) obs: 1.691 / Num. unique obs: 1628 / CC1/2: 0.866

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
Cootmodel building
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8HNT

8hnt


Resolution: 3.41→44.62 Å / SU ML: 0.407 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.6466
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2815 1177 4.88 %
Rwork0.2576 22954 -
obs0.2588 24131 73.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 56.73 Å2
Refinement stepCycle: LAST / Resolution: 3.41→44.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6063 3195 0 0 9258
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00279737
X-RAY DIFFRACTIONf_angle_d0.614213888
X-RAY DIFFRACTIONf_chiral_restr0.03631701
X-RAY DIFFRACTIONf_plane_restr0.00641228
X-RAY DIFFRACTIONf_dihedral_angle_d16.05032585
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.41-3.560.2841330.2549757X-RAY DIFFRACTION19.62
3.56-3.750.2824590.24461210X-RAY DIFFRACTION31.43
3.75-3.990.3185920.26722005X-RAY DIFFRACTION51.8
3.99-4.290.2921810.25943395X-RAY DIFFRACTION87.71
4.3-4.730.26612020.26063873X-RAY DIFFRACTION99.93
4.73-5.410.31671770.25763911X-RAY DIFFRACTION100
5.41-6.810.29892080.27423921X-RAY DIFFRACTION100
6.81-44.620.24962250.24443882X-RAY DIFFRACTION96.21

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