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- EMDB-32385: Structure of Cas7-11 in complex with guide RNA and target RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-32385
TitleStructure of Cas7-11 in complex with guide RNA and target RNA
Map dataSharpened map
Sample
  • Complex: Cas7-11 in complex with a crRNA and its target RNA
    • Complex: crRNA and target RNA
      • RNA: crRNA (39-MER)
      • RNA: tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*AP*CP*AP*AP*AP*G)-3')
    • Complex: Cas7-11
      • Protein or peptide: CRISPR-associated RAMP family protein
  • Ligand: ZINC ION
Function / homologyCRISPR type III-associated protein / RAMP superfamily / defense response to virus / CRISPR-associated RAMP family protein
Function and homology information
Biological speciesEscherichia coli (E. coli) / Desulfonema ishimotonii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.45 Å
AuthorsKato K / Okazaki S / Isayama Y / Nishizawa T / Nishimasu H
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Cell / Year: 2022
Title: Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.
Authors: Kazuki Kato / Wenyuan Zhou / Sae Okazaki / Yukari Isayama / Tomohiro Nishizawa / Jonathan S Gootenberg / Omar O Abudayyeh / Hiroshi Nishimasu /
Abstract: The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and ...The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.
History
DepositionDec 14, 2021-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateJul 6, 2022-
Current statusJul 6, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32385.png

Downloads & links

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Map

FileDownload / File: emd_32385.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-3.9692812 - 6.1255603
Average (Standard dev.)-0.005897766 (±0.104933836)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 424.96 Å
α=β=γ: 90.0 °

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Supplemental data

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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 and target RNA
      • RNA: crRNA (39-MER)
      • RNA: tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*AP*CP*AP*AP*AP*G)-3')
    • Complex: Cas7-11
      • Protein or peptide: CRISPR-associated RAMP family protein
  • 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-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: crRNA and target RNA

SupramoleculeName: crRNA and target RNA / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Cas7-11

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

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Macromolecule #1: crRNA (39-MER)

MacromoleculeName: crRNA (39-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 12.502379 KDa
SequenceString:
GUGAUGUCAC GGAACCUUUG UUGUCUUCGA CAUGGGUAA

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Macromolecule #2: tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*...

MacromoleculeName: tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*AP*CP*AP*AP*AP*G)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 7.377513 KDa
SequenceString:
UACCCAUGUC GAAGACAACA AAG

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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: 185.609875 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 AAKKKFDDSP 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 HHHHHHH

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / 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
GridMaterial: GOLD
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: 2.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 127452

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