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- EMDB-60819: Cryo-EM Structure of CRISPR -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-60819
TitleCryo-EM Structure of CRISPR
Map data
Sample
  • Complex: cas6f-7cas7f complex with RNA
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: CRISPR-associated endonuclease Cas6f/Csy4
    • RNA: RNA (65-MER)
KeywordsRNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas6f/Csy4 / CRISPR-associated protein Csy3
Similarity search - Component
Biological speciesPectobacterium atrosepticum SCRI1043 (bacteria) / Thiocystis violascens DSM 198 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGao X / Cui S / Zhu H / Zhu K / Shang K
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2025
Title: RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.
Authors: Xiaopan Gao / Kaixiang Zhu / Weihe Zhang / Lin Wang / Linyue Wang / Lei Hua / Tongxin Niu / Bo Qin / Xia Yu / Hongtao Zhu / Sheng Cui /
Abstract: RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of ...RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.
History
DepositionJul 16, 2024-
Header (metadata) releaseJan 14, 2026-
Map releaseJan 14, 2026-
UpdateJan 14, 2026-
Current statusJan 14, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_60819.png

Downloads & links

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Map

FileDownload / File: emd_60819.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 480 pix.
= 388.8 Å		0.81 Å/pix.
x 480 pix.
= 388.8 Å		0.81 Å/pix.
x 480 pix.
= 388.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.6
Minimum - Maximum-4.3442593 - 10.916821000000001
Average (Standard dev.)0.0006332702 (±0.35315165)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 388.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_60819_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_60819_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cas6f-7cas7f complex with RNA

EntireName: cas6f-7cas7f complex with RNA
Components
  • Complex: cas6f-7cas7f complex with RNA
    • Protein or peptide: CRISPR-associated protein Csy3
    • Protein or peptide: CRISPR-associated endonuclease Cas6f/Csy4
    • RNA: RNA (65-MER)

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Supramolecule #1: cas6f-7cas7f complex with RNA

SupramoleculeName: cas6f-7cas7f complex with RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)

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Macromolecule #1: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Molecular weightTheoretical: 36.948512 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKAATTLKT ASVLAFERKL ANSDALMYAG NWAQQDNWTA IAIQEKSVRG TISNRLKNAL TSDPAKLDAE IQKANLQKVD VAALPFGAD TLKIVFTLRV LGNLAQPSVC NDQDYQTALG DIITGYAQEQ GFSTLAARYA ENIANGRFLW RNRVGAEAIR V VVTKKGER ...String:
MAKAATTLKT ASVLAFERKL ANSDALMYAG NWAQQDNWTA IAIQEKSVRG TISNRLKNAL TSDPAKLDAE IQKANLQKVD VAALPFGAD TLKIVFTLRV LGNLAQPSVC NDQDYQTALG DIITGYAQEQ GFSTLAARYA ENIANGRFLW RNRVGAEAIR V VVTKKGER SWEFNGEDYS LRQFSQPAGD LAALTQAIEK GLAGDASALF TVEAYVQLGN GQEVFPSQEL VLDEKARNGK SK ILYQVND VAAIHSQKIG NALRTIDDWY PAADEAGPIA VEPYGSVTSR GKAYRQPREK MDFYTLLDNW VIKGDVPMPE QQH YVIATL IRGGVFGEKG E

UniProtKB: CRISPR-associated protein Csy3

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Macromolecule #2: CRISPR-associated endonuclease Cas6f/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6f/Csy4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Molecular weightTheoretical: 20.487529 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MDHYIDIRVQ PDPEFTASQL LNALFAKLHR VLGQLANGKI GISFPEVGKT LGECLRLHGT EDALSTLEKT SWLKGLRDYT QVSECKVVP NGVKFRTVRR VQLKSSAERL RRRSVSKGWL TAAEAAARIP DAVEKRSALP FVQIKSLSNG QMFFVFVEHG P LQNAPTAG RFSSYGLSTE ATVPWF

UniProtKB: CRISPR-associated endonuclease Cas6f/Csy4

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Macromolecule #3: RNA (65-MER)

MacromoleculeName: RNA (65-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Thiocystis violascens DSM 198 (bacteria)
Molecular weightTheoretical: 20.910402 KDa
SequenceString:
UUGCGCGACA GUAGAGACAG GGCGCCGCCU UGUCUGAUGC UCUCGUUCAC UGCCGGAUAG GCAGC

GENBANK: GENBANK: CP003154.1

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 30 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 2500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 4752
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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