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- EMDB-33676: CryoEM structure of type III-E CRISPR gRAMP-crRNA binary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33676
TitleCryoEM structure of type III-E CRISPR gRAMP-crRNA binary complex
Map data
Sample
  • Complex: Type III-E CRISPR gRAMP-crRNA binary complex
    • Complex: gRAMP
      • Protein or peptide: RAMP superfamily protein
    • Complex: crRNA
      • RNA: crRNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsNuclease / STRUCTURAL PROTEIN-RNA COMPLEX
Function / homology: / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / RNA binding / RAMP superfamily protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsZhang JT / Cui N / Huang HD / Jia N
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase.
Authors: Ning Cui / Jun-Tao Zhang / Zhuolin Li / Xiao-Yu Liu / Chongyuan Wang / Hongda Huang / Ning Jia /
Abstract: The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. ...The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMP and gRAMP-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions -1 and -2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.
History
DepositionJun 22, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33676.png

Downloads & links

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Map

FileDownload / File: emd_33676.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.2
Minimum - Maximum-7.522153 - 11.686814
Average (Standard dev.)0.000019519177 (±0.24749912)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33676_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_33676_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_33676_half_map_2.map
Projections & Slices
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Sample components

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Entire : Type III-E CRISPR gRAMP-crRNA binary complex

EntireName: Type III-E CRISPR gRAMP-crRNA binary complex
Components
  • Complex: Type III-E CRISPR gRAMP-crRNA binary complex
    • Complex: gRAMP
      • Protein or peptide: RAMP superfamily protein
    • Complex: crRNA
      • RNA: crRNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Type III-E CRISPR gRAMP-crRNA binary complex

SupramoleculeName: Type III-E CRISPR gRAMP-crRNA binary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)

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Supramolecule #2: gRAMP

SupramoleculeName: gRAMP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)

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Supramolecule #3: crRNA

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

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Macromolecule #1: RAMP superfamily protein

MacromoleculeName: RAMP superfamily protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 198.652688 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHKSN DMNITVELTF FEPYRLVEWF DWDARKKSHS AMRGQAFAQW TWKGKGRTAG KSFITGTLVR SAVIKAVEEL LSLNNGKWE GVPCCNGSFQ TDESKGKKPS FLRKRHTLQW QANNKNICDK EEACPFCILL GRFDNAGKVH ERNKDYDIHF S NFDLDHKQ ...String:
MHHHHHHKSN DMNITVELTF FEPYRLVEWF DWDARKKSHS AMRGQAFAQW TWKGKGRTAG KSFITGTLVR SAVIKAVEEL LSLNNGKWE GVPCCNGSFQ TDESKGKKPS FLRKRHTLQW QANNKNICDK EEACPFCILL GRFDNAGKVH ERNKDYDIHF S NFDLDHKQ EKNDLRLVDI ASGRILNRVD FDTGKAKDYF RTWEADYETY GTYTGRITLR NEHAKKLLLA SLGFVDKLCG AL CRIEVIK KSESPLPSDT KEQSYTKDDT VEVLSEDHND ELRKQAEVIV EAFKQNDKLE KIRILADAIR TLRLHGEGVI EKD ELPDGK EERDKGHHLW DIKVQGTALR TKLKELWQSN KDIGWRKFTE MLGSNLYLIY KKETGGVSTR FRILGDTEYY SKAH DSEGS DLFIPVTPPE GIETKEWIIV GRLKAATPFY FGVQQPSDSI PGKEKKSEDS LVINEHTSFN ILLDKENRYR IPRSA LRGA LRRDLRTAFG SGCNVSLGGQ ILCNCKVCIE MRRITLKDSV SDFSEPPEIR YRIAKNPGTA TVEDGSLFDI EVGPEG LTF PFVLRYRGHK FPEQLSSVIR YWEENDGKNG MAWLGGLDST GKGRFALKDI KIFEWDLNQK INEYIKERGM RGKEKEL LE MGESSLPDGL IPYKFFEERE CLFPYKENLK PQWSEVQYTI EVGSPLLTAD TISALTEPGN RDAIAYKKRV YNDGNNAI E PEPRFAVKSE THRGIFRTAV GRRTGDLGKE DHEDCTCDMC IIFGNEHESS KIRFEDLELI NGNEFEKLEK HIDHVAIDR FTGGALDKAK FDTYPLAGSP KKPLKLKGRF WIKKGFSGDH KLLITTALSD IRDGLYPLGS KGGVGYGWVA GISIDDNVPD DFKEMINKT EMPLPEEVEE SNNGPINNDY VHPGHQSPKQ DHKNKNIYYP HYFLDSGSKV YREKDIITHE EFTEELLSGK I NCKLETLT PLIIPDTSDE NGLKLQGNKP GHKNYKFFNI NGELMIPGSE LRGMLRTHFE ALTKSCFAIF GEDSTLSWRM NA DEKDYKI DSNSIRKMES QRNPKYRIPD ELQKELRNSG NGLFNRLYTS ERRFWSDVSN KFENSIDYKR EILRCAGRPK NYK GGIIRQ RKDSLMAEEL KVHRLPLYDN FDIPDSAYKA NDHCRKSATC STSRGCRERF TCGIKVRDKN RVFLNAANNN RQYL NNIKK SNHDLYLQYL KGEKKIRFNS KVITGSERSP IDVIAELNER GRQTGFIKLS GLNNSNKSQG NTGTTFNSGW DRFEL NILL DDLETRPSKS DYPRPRLLFT KDQYEYNITK RCERVFEIDK GNKTGYPVDD QIKKNYEDIL DSYDGIKDQE VAERFD TFT RGSKLKVGDL VYFHIDGDNK IDSLIPVRIS RKCASKTLGG KLDKALHPCT GLSDGLCPGC HLFGTTDYKG RVKFGFA KY ENGPEWLITR GNNPERSLTL GVLESPRPAF SIPDDESEIP GRKFYLHHNG WRIIRQKQLE IRETVQPERN VTTEVMDK G NVFSFDVRFE NLREWELGLL LQSLDPGKNI AHKLGKGKPY GFGSVKIKID SLHTFKINSN NDKIKRVPQS DIREYINKG YQKLIEWSGN NSIQKGNVLP QWHVIPHIDK LYKLLWVPFL NDSKLEPDVR YPVLNEESKG YIEGSDYTYK KLGDKDNLPY KTRVKGLTT PWSPWNPFQV IAEHEEQEVN VTGSRPSVTD KIERDGKMV

UniProtKB: RAMP superfamily protein

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 35.43209 KDa
SequenceString:
GUUAUGAAAC AAGAGAAGGA CUUAAUGUCA CGGUACCCAA UUUUCUGCCC CGGACUCCAC GGCUGUUACU AGAGGUUAUG AAACAAGAG AAGGACUUAA UGUCACGGUA C

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 227997
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT

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