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- EMDB-34218: Structure of the Cas7-11-Csx29-guide RNA-target RNA (non-matching... -

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Basic information

Entry
Database: EMDB / ID: EMD-34218
TitleStructure of the Cas7-11-Csx29-guide RNA-target RNA (non-matching PFS) complex
Map data
Sample
  • Complex: Cas7-11 in complex with a crRNA and its target RNA
    • Complex: crRNA
      • RNA: crRNA
    • Complex: target RNA with 3'-tag
      • RNA: target RNA with non-matching PFS
    • Complex: Cas7-11, Csx29
      • Protein or peptide: CRISPR-associated RAMP family protein
      • Protein or peptide: CHAT domain-containing protein
  • Ligand: CYTIDINE-5'-MONOPHOSPHATE
  • Ligand: ADENOSINE-5'-MONOPHOSPHATE
  • Ligand: ZINC ION
Function / homologyCHAT domain / CHAT domain / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / CRISPR-associated RAMP family protein / CHAT domain-containing protein
Function and homology information
Biological speciesDesulfonema ishimotonii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsKato K / Okazaki S / Ishikawa J / Isayama Y / Nishizawa T / Nishimasu H
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Science / Year: 2022
Title: RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.
Authors: Kazuki Kato / Sae Okazaki / Cian Schmitt-Ulms / Kaiyi Jiang / Wenyuan Zhou / Junichiro Ishikawa / Yukari Isayama / Shungo Adachi / Tomohiro Nishizawa / Kira S Makarova / Eugene V Koonin / ...Authors: Kazuki Kato / Sae Okazaki / Cian Schmitt-Ulms / Kaiyi Jiang / Wenyuan Zhou / Junichiro Ishikawa / Yukari Isayama / Shungo Adachi / Tomohiro Nishizawa / Kira S Makarova / Eugene V Koonin / Omar O Abudayyeh / Jonathan S Gootenberg / Hiroshi Nishimasu /
Abstract: The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11- ...The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.
History
DepositionSep 4, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateDec 7, 2022-
Current statusDec 7, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34218.png

Downloads & links

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Map

FileDownload / File: emd_34218.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.8408897 - 5.257334
Average (Standard dev.)0.00034972848 (±0.11566863)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34218_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34218_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_34218_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 : Cas7-11 in complex with a crRNA and its target RNA

EntireName: Cas7-11 in complex with a crRNA and its target RNA
Components
  • Complex: Cas7-11 in complex with a crRNA and its target RNA
    • Complex: crRNA
      • RNA: crRNA
    • Complex: target RNA with 3'-tag
      • RNA: target RNA with non-matching PFS
    • Complex: Cas7-11, Csx29
      • Protein or peptide: CRISPR-associated RAMP family protein
      • Protein or peptide: CHAT domain-containing protein
  • Ligand: CYTIDINE-5'-MONOPHOSPHATE
  • Ligand: ADENOSINE-5'-MONOPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: Cas7-11 in complex with a crRNA and its target RNA

SupramoleculeName: Cas7-11 in complex with a crRNA and its target RNA / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Desulfonema ishimotonii (bacteria)

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Supramolecule #2: crRNA

SupramoleculeName: crRNA / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: target RNA with 3'-tag

SupramoleculeName: target RNA with 3'-tag / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Desulfonema ishimotonii (bacteria)

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Supramolecule #4: Cas7-11, Csx29

SupramoleculeName: Cas7-11, Csx29 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4

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Macromolecule #1: crRNA

MacromoleculeName: crRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 30.532891 KDa
SequenceString:
GGUUGGAAAG CCGGUUUUCU UUGAUGUCAC GGAACCUUUG UUGUCUUCGA CAUGGGUAAU GGUUGGAAAG CCGGUUUUCU UUGAUGUCA CGGAAC

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Macromolecule #2: target RNA with non-matching PFS

MacromoleculeName: target RNA with non-matching PFS / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 10.662481 KDa
SequenceString:
GGAUUACCCA UGUCGAAGAC AACAAAGCAA GGC

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Macromolecule #3: CRISPR-associated RAMP family protein

MacromoleculeName: CRISPR-associated RAMP family protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 185.60375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTTTMKISIE FLEPFRMTKW QESTRRNKNN KEFVRGQAFA RWHRNKKDNT KGRPYITGTL LRSAVIRSAE NLLTLSDGKI SEKTCCPGK FDTEDKDRLL QLRQRSTLRW TDKNPCPDNA ETYCPFCELL GRSGNDGKKA EKKDWRFRIH FGNLSLPGKP D FDGPKAIG ...String:
MTTTMKISIE FLEPFRMTKW QESTRRNKNN KEFVRGQAFA RWHRNKKDNT KGRPYITGTL LRSAVIRSAE NLLTLSDGKI SEKTCCPGK FDTEDKDRLL QLRQRSTLRW TDKNPCPDNA ETYCPFCELL GRSGNDGKKA EKKDWRFRIH FGNLSLPGKP D FDGPKAIG SQRVLNRVDF KSGKAHDFFK AYEVDHTRFP RFEGEITIDN KVSAEARKLL CDSLKFTDRL CGALCVIRFD EY TPAADSG KQTENVQAEP NANLAEKTAE QIISILDDNK KTEYTRLLAD AIRSLRRSSK LVAGLPKDHD GKDDHYLWDI GKK KKDENS VTIRQILTTS ADTKELKNAG KWREFCEKLG EALYLKSKDM SGGLKITRRI LGDAEFHGKP DRLEKSRSVS IGSV LKETV VCGELVAKTP FFFGAIDEDA KQTDLQVLLT PDNKYRLPRS AVRGILRRDL QTYFDSPCNA ELGGRPCMCK TCRIM RGIT VMDARSEYNA PPEIRHRTRI NPFTGTVAEG ALFNMEVAPE GIVFPFQLRY RGSEDGLPDA LKTVLKWWAE GQAFMS GAA STGKGRFRME NAKYETLDLS DENQRNDYLK NWGWRDEKGL EELKKRLNSG LPEPGNYRDP KWHEINVSIE MASPFIN GD PIRAAVDKRG TDVVTFVKYK AEGEEAKPVC AYKAESFRGV IRSAVARIHM EDGVPLTELT HSDCECLLCQ IFGSEYEA G KIRFEDLVFE SDPEPVTFDH VAIDRFTGGA ADKKKFDDSP LPGSPARPLM LKGSFWIRRD VLEDEEYCKA LGKALADVN NGLYPLGGKS AIGYGQVKSL GIKGDDKRIS RLMNPAFDET DVAVPEKPKT DAEVRIEAEK VYYPHYFVEP HKKVEREEKP CGHQKFHEG RLTGKIRCKL ITKTPLIVPD TSNDDFFRPA DKEARKEKDE YHKSYAFFRL HKQIMIPGSE LRGMVSSVYE T VTNSCFRI FDETKRLSWR MDADHQNVLQ DFLPGRVTAD GKHIQKFSET ARVPFYDKTQ KHFDILDEQE IAGEKPVRMW VK RFIKRLS LVDPAKHPQK KQDNKWKRRK EGIATFIEQK NGSYYFNVVT NNGCTSFHLW HKPDNFDQEK LEGIQNGEKL DCW VRDSRY QKAFQEIPEN DPDGWECKEG YLHVVGPSKV EFSDKKGDVI NNFQGTLPSV PNDWKTIRTN DFKNRKRKNE PVFC CEDDK GNYYTMAKYC ETFFFDLKEN EEYEIPEKAR IKYKELLRVY NNNPQAVPES VFQSRVAREN VEKLKSGDLV YFKHN EKYV EDIVPVRISR TVDDRMIGKR MSADLRPCHG DWVEDGDLSA LNAYPEKRLL LRHPKGLCPA CRLFGTGSYK GRVRFG FAS LENDPEWLIP GKNPGDPFHG GPVMLSLLER PRPTWSIPGS DNKFKVPGRK FYVHHHAWKT IKDGNHPTTG KAIEQSP NN RTVEALAGGN SFSFEIAFEN LKEWELGLLI HSLQLEKGLA HKLGMAKSMG FGSVEIDVES VRLRKDWKQW RNGNSEIP N WLGKGFAKLK EWFRDELDFI ENLKKLLWFP EGDQAPRVCY PMLRKKDDPN GNSGYEELKD GEFKKEDRQK KLTTPWTPW ASSGLVPRGS HHHHHH

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Macromolecule #4: CHAT domain-containing protein

MacromoleculeName: CHAT domain-containing protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 88.311875 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSNPIRDIQD RLKTAKFDNK DDMMNLASSL YKYEKQLMDS SEATLCQQGL SNRPNSFSQL SQFRDSDIQS KAGGQTGKFW QNEYEACKN FQTHKERRET LEQIIRFLQN GAEEKDADDL LLKTLARAYF HRGLLYRPKG FSVPARKVEA MKKAIAYCEI I LDKNEEES ...String:
MSNPIRDIQD RLKTAKFDNK DDMMNLASSL YKYEKQLMDS SEATLCQQGL SNRPNSFSQL SQFRDSDIQS KAGGQTGKFW QNEYEACKN FQTHKERRET LEQIIRFLQN GAEEKDADDL LLKTLARAYF HRGLLYRPKG FSVPARKVEA MKKAIAYCEI I LDKNEEES EALRIWLYAA MELRRCGEEY PENFAEKLFY LANDGFISEL YDIRLFLEYT EREEDNNFLD MILQENQDRE RL FELCLYK ARACFHLNQL NDVRIYGESA IDNAPGAFAD PFWDELVEFI RMLRNKKSEL WKEIAIKAWD KCREKEMKVG NNI YLSWYW ARQRELYDLA FMAQDGIEKK TRIADSLKSR TTLRIQELNE LRKDAHRKQN RRLEDKLDRI IEQENEARDG AYLR RNPPC FTGGKREEIP FARLPQNWIA VHFYLNELES HEGGKGGHAL IYDPQKAEKD QWQDKSFDYK ELHRKFLEWQ ENYIL NEEG SADFLVTLCR EIEKAMPFLF KSEVIPEDRP VLWIPHGFLH RLPLHAAMKS GNNSNIEIFW ERHASRYLPA WHLFDP APY SREESSTLLK NFEEYDFQNL ENGEIEVYAP SSPKKVKEAI RENPAILLLL CHGEADMTNP FRSCLKLKNK DMTIFDL LT VEDVRLSGSR ILLGACESDM VPPLEFSVDE HLSVSGAFLS HKAGEIVAGL WTVDSEKVDE CYSYLVEEKD FLRNLQEW Q MAETENFRSE NDSSLFYKIA PFRIIGFPAE

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Macromolecule #5: CYTIDINE-5'-MONOPHOSPHATE

MacromoleculeName: CYTIDINE-5'-MONOPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: C
Molecular weightTheoretical: 323.197 Da
Chemical component information

ChemComp-C:
CYTIDINE-5'-MONOPHOSPHATE / Cytidine monophosphate

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Macromolecule #6: ADENOSINE-5'-MONOPHOSPHATE

MacromoleculeName: ADENOSINE-5'-MONOPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: A
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-A:
ADENOSINE-5'-MONOPHOSPHATE / Adenosine monophosphate

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 76.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71543

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