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- EMDB-33958: Cryo-EM structure of Cas7-11-crRNA bound to target RNA-3 -

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Basic information

Entry
Database: EMDB / ID: EMD-33958
TitleCryo-EM structure of Cas7-11-crRNA bound to target RNA-3
Map data
Sample
  • Complex: The complex of Cas7-11-crRNA bound to target RNA-3
    • Protein or peptide: CRISPR-associated RAMP family protein
    • RNA: crRNA (38-MER)
    • RNA: Target RNA-3 (28-MER)
Function / homologyCRISPR type III-associated protein / RAMP superfamily / defense response to virus / CRISPR-associated RAMP family protein
Function and homology information
Biological speciesDesulfonema ishimotonii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsHuo Y / Dong Q / Zhao H / Jiang T
Funding support China, 1 items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37010301 China
CitationJournal: Nat Microbiol / Year: 2023
Title: Cryo-EM structure and protease activity of the type III-E CRISPR-Cas effector.
Authors: Yangao Huo / Hongshen Zhao / Qinghua Dong / Tao Jiang /
Abstract: The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here ...The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here we present four cryo-EM structures of Desulfonema ishimotonii Cas7-11-crRNA complex in pre-target and target RNA-bound states, and the cryo-EM structure of DiCas7-11-crRNA bound to its accessory protein DiCsx29. These data reveal structural elements for pre-crRNA processing, target RNA cleavage and regulation. Moreover, a 3' seed region of crRNA is involved in regulating RNA cleavage activity of DiCas7-11-crRNA-Csx29. Our analysis also shows that both the minimal mismatch of 4 nt to the 5' handle of crRNA and the minimal matching of the first 12 nt of the spacer by the target RNA are essential for triggering the protease activity of DiCas7-11-crRNA-Csx29 towards DiCsx30. Taken together, we propose that target RNA recognition and cleavage regulate and fine-tune the protease activity of DiCas7-11-crRNA-Csx29, thus preventing auto-immune responses.
History
DepositionJul 30, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33958.png

Downloads & links

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Map

FileDownload / File: emd_33958.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0157
Minimum - Maximum-0.110937506 - 0.19337678
Average (Standard dev.)2.1771893e-05 (±0.003505668)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33958_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_33958_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 : The complex of Cas7-11-crRNA bound to target RNA-3

EntireName: The complex of Cas7-11-crRNA bound to target RNA-3
Components
  • Complex: The complex of Cas7-11-crRNA bound to target RNA-3
    • Protein or peptide: CRISPR-associated RAMP family protein
    • RNA: crRNA (38-MER)
    • RNA: Target RNA-3 (28-MER)

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Supramolecule #1: The complex of Cas7-11-crRNA bound to target RNA-3

SupramoleculeName: The complex of Cas7-11-crRNA bound to target RNA-3 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Desulfonema ishimotonii (bacteria)

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Macromolecule #1: CRISPR-associated RAMP family protein

MacromoleculeName: CRISPR-associated RAMP family protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 183.933 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 A

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Macromolecule #2: crRNA (38-MER)

MacromoleculeName: crRNA (38-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 15.094032 KDa
SequenceString:
UUGAUGUCAC GGAACAGGAA CUUGAACAAC AUCGUUACUA ACGAGCU

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Macromolecule #3: Target RNA-3 (28-MER)

MacromoleculeName: Target RNA-3 (28-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 15.018855 KDa
SequenceString:
AGCUCGUUAG UAACGAUGUU GUUCAAGUUC CUCAAGGCAC UGUAGUU

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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: 7.5
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: 2.5 µm / Nominal defocus min: 1.3 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 926347

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