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- EMDB-28065: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30 -

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Basic information

Entry
Database: EMDB / ID: EMD-28065
TitleCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
Map dataCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Cas7-11
Sample
  • Complex: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
    • Protein or peptide: Csx30
    • Protein or peptide: Cas7-11
    • Protein or peptide: Csx29
    • RNA: crRNA
    • RNA: DR-mismatched target RNA
  • Ligand: ZINC ION
KeywordsCRISPR / endonuclease / endopeptidase / RNA BINDING PROTEIN-RNA complex
Function / homologyCHAT domain / CHAT domain / : / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / CRISPR-associated RAMP family protein / Uncharacterized protein / CHAT domain-containing protein
Function and homology information
Biological speciesDesulfonema ishimotonii (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.53 Å
AuthorsDemircioglu FE / Wilkinson ME / Strecker J / Li D / Faure G / Macrae RK / Zhang F
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1DP1-HL141201 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2R01HG009761-05 United States
CitationJournal: Science / Year: 2022
Title: RNA-activated protein cleavage with a CRISPR-associated endopeptidase.
Authors: Jonathan Strecker / F Esra Demircioglu / David Li / Guilhem Faure / Max E Wilkinson / Jonathan S Gootenberg / Omar O Abudayyeh / Hiroshi Nishimasu / Rhiannon K Macrae / Feng Zhang /
Abstract: In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have ...In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.
History
DepositionSep 7, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28065.png

Downloads & links

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Map

FileDownload / File: emd_28065.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Cas7-11
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 320 pix.
= 318.24 Å		0.99 Å/pix.
x 320 pix.
= 318.24 Å		0.99 Å/pix.
x 320 pix.
= 318.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9945 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.054282203 - 0.14339264
Average (Standard dev.)0.00004175599 (±0.003667266)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 318.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28065_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cas7-11 in complex with DR-mismatched target RNA, Csx29...

Fileemd_28065_half_map_1.map
AnnotationCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cas7-11 in complex with DR-mismatched target RNA, Csx29...

Fileemd_28065_half_map_2.map
AnnotationCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, half map 2
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 DR-mismatched target RNA, Csx29 and Csx30

EntireName: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
Components
  • Complex: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
    • Protein or peptide: Csx30
    • Protein or peptide: Cas7-11
    • Protein or peptide: Csx29
    • RNA: crRNA
    • RNA: DR-mismatched target RNA
  • Ligand: ZINC ION

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Supramolecule #1: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30

SupramoleculeName: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Desulfonema ishimotonii (bacteria)

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Macromolecule #1: Csx30

MacromoleculeName: Csx30 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 62.688184 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNTTTYNTTT DALLEWGKVY FQKEDFSEFL DNLEAYISDA GDSLKDELES GVEKLVLGIK SAEAVIFGEA VIGTTPENEA WYDAEESFL TLDCAVWLSQ ALDRVVRRQD ASLADSLIAR LDEAINRVAE KLYADNLSPL RFSSLNEIRR SALEATDEKY H YLFPWHGA ...String:
MNTTTYNTTT DALLEWGKVY FQKEDFSEFL DNLEAYISDA GDSLKDELES GVEKLVLGIK SAEAVIFGEA VIGTTPENEA WYDAEESFL TLDCAVWLSQ ALDRVVRRQD ASLADSLIAR LDEAINRVAE KLYADNLSPL RFSSLNEIRR SALEATDEKY H YLFPWHGA ACDVDENILL ILTEEYHLIG ADKAGANLSE ELRGDLPFIF AELERDEVLR AYVEKENALS LALENTMREH WA FGLLEAA RDEGYNHPYP ADVGMRIHQV ARAVFSQTNL SPAERLAVAI AGACFTPEIS EDRRLEILLD CEERVCEIEA PTG DDTSVR VIKDLKALAD HRVRHEIPAE SLVSLWFEQI EAAGTDFDTK TPMDELVLRM LSDNVITLSV DRKAASQTET DDVK PQKGK IIPFPVPDIA NDEVEYQKAV GGGSANDSKV KFPGLLEIQG CRDGDKAILL EDTDDAAANH RKLFSILKAG KLNSA FFIQ SDDGEWVESE SKPTMEDNRI ILHDSHHSSF VWILDTGSMQ LRQSVKCVKD ALNKKTGSAK KLKPKTMIVW VTIPQE G

UniProtKB: Uncharacterized protein, Uncharacterized protein

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Macromolecule #2: Cas7-11

MacromoleculeName: Cas7-11 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 183.844984 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 KQTALQVLLT 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 TAVVTFVKYK 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 A

UniProtKB: CRISPR-associated RAMP family protein

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Macromolecule #3: Csx29

MacromoleculeName: Csx29 / type: protein_or_peptide / ID: 3 / 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

UniProtKB: CHAT domain-containing protein

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Macromolecule #4: crRNA

MacromoleculeName: crRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 12.134133 KDa
SequenceString:
UUGAUGUCAC GGAACCUUUG UUGUCUUCGA CAUGGGUA

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Macromolecule #5: DR-mismatched target RNA

MacromoleculeName: DR-mismatched target RNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.931381 KDa
SequenceString:
UACCCAUGUC GAAGACAACA AAGUAUUU

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 65733
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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