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- EMDB-37448: Cryo-EM structure of Cas13h1-crRNA binary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-37448
TitleCryo-EM structure of Cas13h1-crRNA binary complex
Map data
Sample
  • Complex: RNA-protein complex
    • Complex: Pro
      • Protein or peptide: Cas13h1
    • Complex: RNA
      • RNA: 66-nt crRNA
  • Ligand: MAGNESIUM ION
KeywordsRNA BINDING PROTEIN-RNA COMPLEX
Biological speciesunidentified (others) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsZhang C
Funding support China, 1 items
OrganizationGrant numberCountry
Not funded China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular mechanism for target RNA recognition and cleavage of Cas13h.
Authors: Fugen Chen / Chendi Zhang / Jialin Xue / Feng Wang / Zhuang Li /
Abstract: RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA ...RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.
History
DepositionSep 13, 2023-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_37448.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.255
Minimum - Maximum-1.2258753 - 1.962766
Average (Standard dev.)0.00083994615 (±0.07654282)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 204.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37448_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_37448_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : RNA-protein complex

EntireName: RNA-protein complex
Components
  • Complex: RNA-protein complex
    • Complex: Pro
      • Protein or peptide: Cas13h1
    • Complex: RNA
      • RNA: 66-nt crRNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: RNA-protein complex

SupramoleculeName: RNA-protein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: unidentified (others)

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Supramolecule #2: Pro

SupramoleculeName: Pro / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Cas13h1

MacromoleculeName: Cas13h1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 130.957328 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDTPSKESSG KIIEKLDELK KATQESIEQR KKFDYRIGKG LADKLGNEKD SYFEKYLRKT VGENIPPKIL VKPKGGWKTE TKGKGKDWG DKITFAAMPQ HHYNTLLAMA FTKAHKVYSD IRGKVEIDAA QYETKAKLIE VEYGKDETRG LSGLEYLMMS K HLFSGKNS ...String:
MDTPSKESSG KIIEKLDELK KATQESIEQR KKFDYRIGKG LADKLGNEKD SYFEKYLRKT VGENIPPKIL VKPKGGWKTE TKGKGKDWG DKITFAAMPQ HHYNTLLAMA FTKAHKVYSD IRGKVEIDAA QYETKAKLIE VEYGKDETRG LSGLEYLMMS K HLFSGKNS NIKLAVKKGE TEILKEYALN EKLIYTVLDE LRNFHSHIFH EPGPVSFKNL YGDEYKPEKK LTEEEWAIAR DW FVNRFND AKEHKLKTLA KVLEREGTTE EKEDAEKVIK TISGYSFEYN NCISREALLF IACMFLRKSD AAYFTKKWTG MKK AEGVFK STQSFFTDNA LKESKSILTL NADLYKYRQI LGVLSTMPAM KTDSLKPFYD FIKINNDSYS EKAEKARSKE EKEK IQAFI IPQRKSSNYT YWFMKYLNDN KLLDGFRIAY YKTPEDRFMY LIHNGLISQD DLENIEDFKT PDEKLKYLRE KGFNL KLKM KQAVGDEKKS LTEIYKETQR NFVFKVPTIE NDNFCVKKLN VFFQTDIEFN GQKISVQLSV SPDFLMKWVF VLLITG EDS IKNAITEKKE KIKDILKKYA EEYYNRCITS NFNEPLMGLE ASKVFPSSLT STVEIDEKID KDKILMRISE KYNELTK FD EENKSRKAPW RFASKRKIDI ILDYVHLVYS DRAFDEKKSV DAMRHEALND MEYMDTFEYL RYYGRYRETE EFKKIFFE D KKLYFSPILK AMKQLDSLEG VFNFAITGFL NYLKGIQSKV TDENTNKYGK VFKVTGKSLT SKIGHHSEMF SVNHCVPQE LIKLNDIKGY MKWKHETKDK LWISDFAFIR NVLESRGGFS NTDYLMKEVM PLITFEKNEK GSIKGNTQMF VALSRNKTNE LMLWEIGKY YWKEATGSEF SRLFKGLEKN TGNKITKAYR FTNPYYTIYQ EDLDIKIQRK DKKGKVIVNS PVYTIKIKPK K FDDEYQYY EQEHIVDYIE NYEPKKGIDG HWHFEELNKK IKDELARYLD DIYLLMTVEK HIVQKDFDKY ASLLKEKDSK LP PYYIGFK RNDIALNKVI FDALKHKGSF SADDLNIYRI NVLHQILQPK REKYIIIRKS LIEYCAENKL LKTM

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Macromolecule #2: 66-nt crRNA

MacromoleculeName: 66-nt crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 21.257666 KDa
SequenceString:
UGCUUCACGU AGGCCUUGGA GCCGUACAUG GUUGUAACAA GCCUAAGUUU GAAAGGUAAA AACAAC

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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: 7.5
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: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 207575
FSC plot (resolution estimation)

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