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- EMDB-27262: gRAMP-TPR-CHAT match PFS target RNA(Craspase) -

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Basic information

Entry
Database: EMDB / ID: EMD-27262
TitlegRAMP-TPR-CHAT match PFS target RNA(Craspase)
Map data
Sample
  • Complex: gRAMP-TPR-CHAT with match PFS target RNA Craspase
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (36-MER)
    • RNA: RNA (5'-R(P*UP*CP*CP*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
    • Protein or peptide: CHAT domain protein
  • Ligand: ZINC ION
  • Ligand: PHOSPHATE IONPhosphate
KeywordsCRISPR / GRAMP / RNA BINDING PROTEIN / Craspase / RNA BINDING PROTEIN-RNA complex
Function / homologyCHAT domain / CHAT domain / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / RAMP superfamily protein / CHAT domain protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHu C / Nam KH / Schuler G / Ke A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118174 United States
CitationJournal: Science / Year: 2022
Title: Craspase is a CRISPR RNA-guided, RNA-activated protease.
Authors: Chunyi Hu / Sam P B van Beljouw / Ki Hyun Nam / Gabriel Schuler / Fran Ding / Yanru Cui / Alicia Rodríguez-Molina / Anna C Haagsma / Menno Valk / Martin Pabst / Stan J J Brouns / Ailong Ke /
Abstract: The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron ...The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.
History
DepositionJun 9, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27262.png

Downloads & links

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Map

FileDownload / File: emd_27262.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.077
Minimum - Maximum-0.574834 - 1.063134
Average (Standard dev.)-0.00072515453 (±0.037601225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_27262_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27262_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27262_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : gRAMP-TPR-CHAT with match PFS target RNA Craspase

EntireName: gRAMP-TPR-CHAT with match PFS target RNA Craspase
Components
  • Complex: gRAMP-TPR-CHAT with match PFS target RNA Craspase
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (36-MER)
    • RNA: RNA (5'-R(P*UP*CP*CP*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
    • Protein or peptide: CHAT domain protein
  • Ligand: ZINC ION
  • Ligand: PHOSPHATE IONPhosphate

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Supramolecule #1: gRAMP-TPR-CHAT with match PFS target RNA Craspase

SupramoleculeName: gRAMP-TPR-CHAT with match PFS target RNA Craspase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)

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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: 145.301531 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MNITVELTFF EPYRLVEWFD WDARKKSHSA MRGQAFAQWT WKGKGRTAGK SFITGTLVRS AVIKAVEELL SLNNGKWEGV PCCNGSFQT DESKGKKPSF LRKRHTLQWQ ANNKNICDKE EACPFCILLG RFDNAGKVHE RNKDYDIHFS NFDLDHKQEK N DLRLVDIA ...String:
MNITVELTFF EPYRLVEWFD WDARKKSHSA MRGQAFAQWT WKGKGRTAGK SFITGTLVRS AVIKAVEELL SLNNGKWEGV PCCNGSFQT DESKGKKPSF LRKRHTLQWQ ANNKNICDKE EACPFCILLG RFDNAGKVHE RNKDYDIHFS NFDLDHKQEK N DLRLVDIA SGRILNRVDF DTGKAKDYFR TWEADYETYG TYTGRITLRN EHAKKLLLAS LGFVDKLCGA LCRIEVIKDH ND ELRKQAE VIVEAFKQND KLEKIRILAD AIRTLRLHGE GVIEKDELPD GKEERDKGHH LWDIKVQGTA LRTKLKELWQ SNK DIGWRK FTEMLGSNLY LIYKKETGGV STEYYSKAHD SEGSDLFIPV TPPEGIETKE WIIVGRLKAA TPFYFGVQQP SDSI PGKEI NEHTSFNILL DKENRYRIPR SALRGALRRD LRTAFGSGCN VSLGGQILCN CKVCIEMRRI TLKDSVSDFS EPPEI RYRI AKNPGTATVE DGSLFDIEVG PEGLTFPFVL RYRGHKFPEQ LSSVIRYWEE NDGKNGMAWL GGLDSTGKGR FALKDI KIF EWDLNQKINE YIKERGMRGK EKELLEMGES SLPDGLIPYK FFEERECLFP YKENLKPQWS EVQYTIEVGS PLLTADT IS ALTEPGNRDA IAYKKRVYND GNNAIEPEPR FAVKSETHRG IFRTAVGRRT GDLGKEDHED CTCDMCIIFG NEHESSKI R FEDLELINGN EFEKLEKHID HVAIDRFTGG ALDKAKFDTY PLAGSPKKPL KLKGRFWIKK GFSGDHKLLI TTALSDIRD GLYPLGSKGG VGYGWVAGIS IDDNVPDDFK EMINKTNNDY VHPGHQSPKQ DHKNKNIYYP HYFLDSGSKV YREKDIITHE EFTEELLSG KINCKLETLT PLIIPDTSDE NGLKLQGNKP GHKNYKFFNI NGELMIPGSE LRGMLRTHFE ALTKSCFAIF G EDSTLSWA SKTLGGKLDK ALHPCTGLSD GLCPGCHLFG TTDYKGRVKF GFAKYENGPE WLITRGNNPE RSLTLGVLES PR PAFSIPD DESEIPGRKF YLHHNGWRII RQKQLEIRET VQPERNVTTE VMDKGNVFSF DVRFENLREW ELGLLLQSLD PGK NIAHKL GKGKPYGFGS VKIKIDSLHT FKINSNNDKI KRVPQSDIRE YINKGYQKLI EWSGNNSIQK GNVLPQWHVI PHID KLYKL LWVPFLNDSK LEPDVRYPVL NEESKGYIEG SDYTYKKLGD KDNLPYKTRV KGLTTPWSPW NPFQV

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Macromolecule #4: CHAT domain protein

MacromoleculeName: CHAT domain protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 76.802992 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: TREDIDRKEA ERLLDEAFNP RTKPVDRKKI INSALKILIG LYKEKKDDLT SASFISIARA YYLVSITILP KGTTIPEKKK EALRKGIEF IDRAINKFNG SILDSQRAFR IKSVLSIEFN RIDREKCDNI KLKNLLNEAV DKGCTDFDTY EWDIQIAIRL C ELGVDMEG ...String:
TREDIDRKEA ERLLDEAFNP RTKPVDRKKI INSALKILIG LYKEKKDDLT SASFISIARA YYLVSITILP KGTTIPEKKK EALRKGIEF IDRAINKFNG SILDSQRAFR IKSVLSIEFN RIDREKCDNI KLKNLLNEAV DKGCTDFDTY EWDIQIAIRL C ELGVDMEG HFDNLIKSNK ANDLQKAKAY YFIKKDDHKA KEHMDKCTAS LKYTPCSHRL WDETVGFIER LKGDSSTLWR DF AIKTYRS CRVQEKETGT LRLRWYWSRH RVLYDMAFLA VKEQADVNVK QAKIKKLAEI SDSLKSRFSL RLSDMEKMPK SDD ESNHEF KKFLDKCVTA YQDGYVIKLL ELTQVPEGWV VVHFYLNKLE GMGNAIVFDK CANSWQYKEF QYKELFEVFL TWQA NYNLY KENAAEHLVT LCKKIGETMP FLFCDNFIPN GKDVLFVPHD FLHRLPLHGS IENKTNGKLF LENHSCCYLP AWSFA SEKE ASTSDEYVLL KNFDQGHFET LQNNQIWGTQ SVKDGASSDD LENIRNNPRL LTILCHGEAN MSNPFRSMLK LANGGI TYL EILNSVKGLK GSQVILGACE TDLVPPLSHY SVATALLLIG AAGVVGTMWK VRSNKTKSLI EWKLENIEYK LNEWQKE TG GAAYKDHPPT FYRSIAFRSI GFPL

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Macromolecule #2: RNA (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 11.423787 KDa
SequenceString:
GACUUAAUGU CACGGUACCC AAUUUUCUGC CCCGGA

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

MacromoleculeName: RNA (5'-R(P*UP*CP*CP*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 6.502928 KDa
SequenceString:
UCCGGGGCAG AAAAUUGGGU

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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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: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 126021

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