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- EMDB-74542: Structure of SpyCas9 in complex with the anti-CRISPR protein AcrIIA26 -

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Basic information

Entry
Database: EMDB / ID: EMD-74542
TitleStructure of SpyCas9 in complex with the anti-CRISPR protein AcrIIA26
Map dataMap with poor HNH density
Sample
  • Complex: Cas9-sgRNA-AcrIIA26 complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • Protein or peptide: AcrIIA26
    • RNA: sgRNA
KeywordsCRISPR / Cas9 / Acr / IMMUNE SYSTEM
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
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria) / Streptococcus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsZheng I / Learn B / Bailey S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM097330 United States
CitationJournal: 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, 2025-
Header (metadata) releaseFeb 4, 2026-
Map releaseFeb 4, 2026-
UpdateFeb 4, 2026-
Current statusFeb 4, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_74542.png

Downloads & links

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Map

FileDownload / File: emd_74542.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap with poor HNH density
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.31255442 - 0.9208798
Average (Standard dev.)0.0030594761 (±0.029853525)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 238.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Combined map sharpened with deepemhancer

Fileemd_74542_additional_1.map
AnnotationCombined map sharpened with deepemhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of the map with poor HNH density

Fileemd_74542_half_map_1.map
AnnotationHalf map B of the map with poor HNH density
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of the map with poor HNH density

Fileemd_74542_half_map_2.map
AnnotationHalf map A of the map with poor HNH density
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas9-sgRNA-AcrIIA26 complex

EntireName: Cas9-sgRNA-AcrIIA26 complex
Components
  • Complex: Cas9-sgRNA-AcrIIA26 complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • Protein or peptide: AcrIIA26
    • RNA: sgRNA

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Supramolecule #1: Cas9-sgRNA-AcrIIA26 complex

SupramoleculeName: Cas9-sgRNA-AcrIIA26 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)
Molecular weightTheoretical: 158.699844 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString: MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: AcrIIA26

MacromoleculeName: AcrIIA26 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus (bacteria)
Molecular weightTheoretical: 21.44926 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString:
MKKLYIQTNQ FANGELQVEN TSYELCDTFK ELYSVASNLV DENTLNFVED NFIEQNYKDE YNGVYENDGD TGEFVGQVFE NKVTEEQFK ELLEQLEITY TEFDPEEELA KCIANKNRKS EFYGNGLKVI AEYLESISHE DALAVVTYYY FYFGFGYEDQ L ISDIKDDQ EDGVKFEHVE RSETI

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Macromolecule #3: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)
Molecular weightTheoretical: 38.697953 KDa
SequenceString:
GGAGAGUCUC UCAGCUGGUA CAGUUUUAGA GCUAUGCUGU UUUGAAAAAA ACAGCAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAA CUUGAAAAAG UGGCACCGAG UCGGUGCUUC G

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 59914
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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