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- EMDB-27138: Cryo-EM structure of guide RNA and target RNA bound Cas7-11 -

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Basic information

Entry
Database: EMDB / ID: EMD-27138
TitleCryo-EM structure of guide RNA and target RNA bound Cas7-11
Map dataCryoEM structure of typeIIIE Cas7-11
Sample
  • Complex: CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii
    • Protein or peptide: CRISPR-associated RAMP family protein
    • RNA: CRISPR RNA (34-MER)
    • RNA: SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*AP*AP*CP*G)-3')
  • 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 speciesDesulfonema ishimotonii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsRai J / Goswami H / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM R01 101343 United States
CitationJournal: Elife / Year: 2022
Title: Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA.
Authors: Hemant N Goswami / Jay Rai / Anuska Das / Hong Li /
Abstract: Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and ...Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.
History
DepositionMay 27, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateNov 2, 2022-
Current statusNov 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27138.png

Downloads & links

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Map

FileDownload / File: emd_27138.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of typeIIIE Cas7-11
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0146
Minimum - Maximum-0.052792046 - 0.09779194
Average (Standard dev.)1.8550476e-05 (±0.0028123718)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-maps1

Fileemd_27138_half_map_1.map
AnnotationHalf-maps1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map2

Fileemd_27138_half_map_2.map
AnnotationHalf-map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii

EntireName: CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii
Components
  • Complex: CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii
    • Protein or peptide: CRISPR-associated RAMP family protein
    • RNA: CRISPR RNA (34-MER)
    • RNA: SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*AP*AP*CP*G)-3')
  • Ligand: ZINC ION

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Supramolecule #1: CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii

SupramoleculeName: CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: CRISPR RNA (34-MER)

MacromoleculeName: CRISPR RNA (34-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 10.837447 KDa
SequenceString:
UUGAUGUCAC GGAACACGUU CUUUGAACCA AGCU

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Macromolecule #3: SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*A...

MacromoleculeName: SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*AP*AP*CP*G)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Molecular weightTheoretical: 5.796516 KDa
SequenceString:
AGCUUGGUUC AAAGAACG

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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: 8
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: 2.2 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (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: OTHER
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 226320

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