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- EMDB-60812: Cryo-EM Structure of csy1-4 with crRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-60812
TitleCryo-EM Structure of csy1-4 with crRNA
Map data
Sample
  • Complex: csy1-4 complex of CRISPR with crRNA
    • Protein or peptide: CRISPR-associated protein Csy2
    • Protein or peptide: CRISPR-associated endonuclease Cas6f/Csy4
    • Protein or peptide: CRISPR-associated protein Csy3
    • RNA: crRNA (87-MER)
    • Protein or peptide: CRISPR-associated protein Csy1
KeywordscrRNA / cas6f / cas7f / cas5f / cas8f / RNA 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 protein Csy1 / CRISPR-associated protein (Cas_Csy1) / 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 Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas6f/Csy4 / CRISPR-associated protein Csy3 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy1
Similarity search - Component
Biological speciesPectobacterium atrosepticum SCRI1043 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 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 15, 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_60812.png

Downloads & links

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Map

FileDownload / File: emd_60812.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 400 pix.
= 300. Å		0.75 Å/pix.
x 400 pix.
= 300. Å		0.75 Å/pix.
x 400 pix.
= 300. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.75 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.0017444838 - 1.9872067
Average (Standard dev.)0.0016978988 (±0.029808475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 300.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_60812_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60812_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 : csy1-4 complex of CRISPR with crRNA

EntireName: csy1-4 complex of CRISPR with crRNA
Components
  • Complex: csy1-4 complex of CRISPR with crRNA
    • Protein or peptide: CRISPR-associated protein Csy2
    • Protein or peptide: CRISPR-associated endonuclease Cas6f/Csy4
    • Protein or peptide: CRISPR-associated protein Csy3
    • RNA: crRNA (87-MER)
    • Protein or peptide: CRISPR-associated protein Csy1

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Supramolecule #1: csy1-4 complex of CRISPR with crRNA

SupramoleculeName: csy1-4 complex of CRISPR with crRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)

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Macromolecule #1: CRISPR-associated protein Csy2

MacromoleculeName: CRISPR-associated protein Csy2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Molecular weightTheoretical: 34.903383 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSTLIILRRI QVENANAIAG LTYGFPAITH FLGFTHALSR KLQASHGLTL EGCGVVSHQH QLHAYGSSWE RSFALTRNPL TKEAKTAAF NEEGRMHMTV SLLIRCDGQI PADTTALCEH LKQQAQCQRL AGGTVIDIER VTVQSLPVDE AETRGVMRRL L PGFVLRDR ...String:
MSTLIILRRI QVENANAIAG LTYGFPAITH FLGFTHALSR KLQASHGLTL EGCGVVSHQH QLHAYGSSWE RSFALTRNPL TKEAKTAAF NEEGRMHMTV SLLIRCDGQI PADTTALCEH LKQQAQCQRL AGGTVIDIER VTVQSLPVDE AETRGVMRRL L PGFVLRDR TSLLHRHFQT LQQAKPQAEM IDAWLDFAAL KMQAERDPSD ETVQWKYLPK PGDGGFLTPL MIGYRAISPL YA PGEVDKT RDPHTPFCFA EAAYGIGEWQ GAHRISDISQ ILWEYDYQNG DYHCRQVADT HSVAEDTSYE FDY

UniProtKB: CRISPR-associated protein Csy2

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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 (E. coli)
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: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Molecular weightTheoretical: 36.948512 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 #5: CRISPR-associated protein Csy1

MacromoleculeName: CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Molecular weightTheoretical: 50.453484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRNGLPEFIL SYINNRKQAK LDAFDKEAEK KRATLSGEAL SVAELELAKA RREIEQKHEV RNWLTDAASR AGQISLVTHA LKFTHSDAK GSSVFNAETV EDATTLSTAT LAQPAIDAVG NAAALDVAKL LQTEHDGDSL VAALQRGDNR ALEALAENPE Q LAQWLTGF ...String:
MRNGLPEFIL SYINNRKQAK LDAFDKEAEK KRATLSGEAL SVAELELAKA RREIEQKHEV RNWLTDAASR AGQISLVTHA LKFTHSDAK GSSVFNAETV EDATTLSTAT LAQPAIDAVG NAAALDVAKL LQTEHDGDSL VAALQRGDNR ALEALAENPE Q LAQWLTGF QQVFTNRQPS SHKLAKQIYF PLANGEYHLL SPLYSSSLAH ALHQRISAVR FGDEAKAIRQ AQRTNQWHDQ LS ISYPNLA VQNMGGTKPQ NISALNSSRS GRSYLLSSAP PQWNSIEKPP QQHESIFRPR GEVDYHTRAT LAQMQRFLLS VKD VENNRD IRQQRLHYLD QLIDQLFFYV ASVQNLPVGW SAESELKRAQ QLWLDPYRAE TDTVFRRERE AGDWQQAVAY EFGR WLNRR LKHENLIFGE VERREWSTAA LFKRRMREME SALKEELA

UniProtKB: CRISPR-associated protein Csy1

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Macromolecule #4: crRNA (87-MER)

MacromoleculeName: crRNA (87-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Molecular weightTheoretical: 28.060721 KDa
SequenceString:
UUCACUGCCG UACAGGCAGC UUAGAAAGCA GAGACUAUCG AUACGGUCUG GACGGAUGCG UUCACUGCCG UACAGGCAGC UUAGAAA

GENBANK: GENBANK: BX950851.1

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
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: 55.0 e/Å2
Electron beamAcceleration voltage: 300 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71806
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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