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- PDB-9zq6: Structure of SpyCas9 in complex with the anti-CRISPR protein AcrIIA26 -

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Basic information

Entry
Database: PDB / ID: 9zq6
TitleStructure of SpyCas9 in complex with the anti-CRISPR protein AcrIIA26
Components
  • AcrIIA26
  • CRISPR-associated endonuclease Cas9/Csn1
  • sgRNA
KeywordsIMMUNE SYSTEM / CRISPR / Cas9 / Acr
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria)
Streptococcus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsZheng, I. / Learn, B. / Bailey, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM097330 United States
Citation
Journal: Biochem J / Year: 2026
Title: Structural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.
Authors: Scott Bailey / Iris Zheng / Brian Learn /
Abstract: CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this ...CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-sgRNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the PAM duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple Acrs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.
#1: Journal: Commun Biol / Year: 2019
Title: SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM.
Authors: Thorsten Wagner / Felipe Merino / Markus Stabrin / Toshio Moriya / Claudia Antoni / Amir Apelbaum / Philine Hagel / Oleg Sitsel / Tobias Raisch / Daniel Prumbaum / Dennis Quentin / Daniel ...Authors: Thorsten Wagner / Felipe Merino / Markus Stabrin / Toshio Moriya / Claudia Antoni / Amir Apelbaum / Philine Hagel / Oleg Sitsel / Tobias Raisch / Daniel Prumbaum / Dennis Quentin / Daniel Roderer / Sebastian Tacke / Birte Siebolds / Evelyn Schubert / Tanvir R Shaikh / Pascal Lill / Christos Gatsogiannis / Stefan Raunser /
Abstract: Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of ...Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.
#2: Journal: Nat Methods / Year: 2020
Title: Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction.
Authors: Ali Punjani / Haowei Zhang / David J Fleet /
Abstract: Cryogenic electron microscopy (cryo-EM) is widely used to study biological macromolecules that comprise regions with disorder, flexibility or partial occupancy. For example, membrane proteins are ...Cryogenic electron microscopy (cryo-EM) is widely used to study biological macromolecules that comprise regions with disorder, flexibility or partial occupancy. For example, membrane proteins are often kept in solution with detergent micelles and lipid nanodiscs that are locally disordered. Such spatial variability negatively impacts computational three-dimensional (3D) reconstruction with existing iterative refinement algorithms that assume rigidity. We introduce non-uniform refinement, an algorithm based on cross-validation optimization, which automatically regularizes 3D density maps during refinement to account for spatial variability. Unlike common shift-invariant regularizers, non-uniform refinement systematically removes noise from disordered regions, while retaining signal useful for aligning particle images, yielding dramatically improved resolution and 3D map quality in many cases. We obtain high-resolution reconstructions for multiple membrane proteins as small as 100 kDa, demonstrating increased effectiveness of cryo-EM for this class of targets critical in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package.
History
DepositionDec 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9/Csn1
B: AcrIIA26
C: sgRNA


Theoretical massNumber of molelcules
Total (without water)218,8473
Polymers218,8473
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158699.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli B (bacteria)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: Protein AcrIIA26


Mass: 21449.260 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus (bacteria) / Production host: Escherichia coli B (bacteria)
#3: RNA chain sgRNA


Mass: 38697.953 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes M1 GAS (bacteria)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cas9-sgRNA-AcrIIA26 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)
Source (recombinant)Organism: Escherichia coli B (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6particle selection
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59914 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 68.61 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003514327
ELECTRON MICROSCOPYf_angle_d0.644919685
ELECTRON MICROSCOPYf_chiral_restr0.03922240
ELECTRON MICROSCOPYf_plane_restr0.00572221
ELECTRON MICROSCOPYf_dihedral_angle_d8.36972607

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