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- EMDB-27260: gRAMP-TPR-CHAT (Craspase) -

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Basic information

Entry
Database: EMDB / ID: EMD-27260
TitlegRAMP-TPR-CHAT (Craspase)
Map data
Sample
  • Complex: gRAMP-TPR-CHAT Craspase
    • Protein or peptide: CHAT domain protein
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (33-MER)
  • Ligand: ZINC ION
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: 2.71 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_27260.png

Downloads & links

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Map

FileDownload / File: emd_27260.map.gz / Format: CCP4 / Size: 46.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.34671497 - 0.8163527
Average (Standard dev.)-0.0011054941 (±0.027315935)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions230230230
Spacing230230230
CellA=B=C: 246.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27260_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_27260_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 : gRAMP-TPR-CHAT Craspase

EntireName: gRAMP-TPR-CHAT Craspase
Components
  • Complex: gRAMP-TPR-CHAT Craspase
    • Protein or peptide: CHAT domain protein
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (33-MER)
  • Ligand: ZINC ION

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Supramolecule #1: gRAMP-TPR-CHAT Craspase

SupramoleculeName: gRAMP-TPR-CHAT Craspase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)

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Macromolecule #1: CHAT domain protein

MacromoleculeName: CHAT domain protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 77.26343 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 YQDGYVILLE LTQVPEGWVV VHFYLNKLEG MGNAIVFDKC ANSWQYKEFQ YKELFEVFLT WQAN YNLYK ENAAEHLVTL CKKIGETMPF LFCDNFIPNG KDVLFVPHDF LHRLPLHGSI ENKTNGKLFL ENHSCCYLPA WSFAS EKEA STSDEYVLLK NFDQGHFETL QNNQIWGTQS VKDGASSDDL ENIRNNPRLL TILCHGEANM SNPFRSMLKL ANGGIT YLE ILNSVKGLKG SQVILGACET DLVPPLSDVM DEHYSVATAL LLIGAAGVVG TMWKVRSNKT KSLIEWKLEN IEYKLNE WQ KETGGAAYKD HPPTFYRSIA FRSIGFPL

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Macromolecule #2: RAMP superfamily protein

MacromoleculeName: RAMP superfamily protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 142.320609 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 NFDLDHDLRL V DIASGRIL ...String:
MNITVELTFF EPYRLVEWFD WDARKKSHSA MRGQAFAQWT WKGKGRTAGK SFITGTLVRS AVIKAVEELL SLNNGKWEGV PCCNGSFQT DESKGKKPSF LRKRHTLQWQ ANNKNICDKE EACPFCILLG RFDNAGKVHE RNKDYDIHFS NFDLDHDLRL V DIASGRIL NRVDFDTGKA KDYFRTWEAD YETYGTYTGR ITLRNEHAKK LLLASLGFVD KLCGALCRIE VIKSEDHNDE LR KQAEVIV EAFKQNDKLE KIRILADAIR TLRLHGEGVI EKDELPDGKE ERDKGHHLWD IKVQGTALRT KLKELWQSNK DIG WRKFTE MLGSNLYLIY KKETGGVSTR FRILGDTEYY SKAHDSEGSD LFIPVTPPEG IETKEWIIVG RLKAATPFYF GVQQ PSDSI PGKEKKSEDS LVINEHTSFN ILLDKENRYR IPRSALRGAL RRDLRTAFGS GCNVSLGGQI LCNCKVCIEM RRITL KDSV SDFSEPPEIR YRIAKNPGTA TVEDGSLFDI EVGPEGLTFP FVLRYRGHKF PEQLSSVIRY WEENDGKNGM AWLGGL DST GKGRFALKDI KIFEWDLNQK INEYIKERGM RGKEKELLEM GESSLPDGLI PYKFFEEREC LFPYKENLKP QWSEVQY TI EVGSPLLTAD TISALTEPGN RDAIAYKKRV YNDGNNAIEP EPRFAVKSET HRGIFRTAVG RRTGDLGKED HEDCTCDM C IIFGNEHESS KIRFEDLELI NGNEFEKLEK HIDHVAIDRF TGGALDKAKF DTYPLAGSPK KPLKLKGRFW IKKGFSGDH KLLITTALSD IRDGLYPLGS KGGVGYGWVA GISIDDNVPD DFKEMINKTY VHPGHQSPKQ DHKNKNIYYP HYFLDSGSKV YREKDIITH EEFTEELLSG KINCKLETLT PLIIPDTSDE NGLKLQGNKP GHKNYKFFNI NGELMIPGSE LRGMLRTHFE A LTKSCFAI FGEGGKLDKA LHPCTGLSDG LCPGCHLFGT TDYKGRVKFG FAKYENGPEW LITRGNNPER SLTLGVLESP RP AFSIPDD ESEIPGRKFY LHHNGWRIIR QKQLEIRETV QPERNVTTEV MDKGNVFSFD VRFENLREWE LGLLLQSLDP GKN IAHKLG KGKPYGFGSV KIKIDSLHTF KIIKRVPQSD IREYINKGYQ KLIEWSLPQW HVIPHIDKLY KLLWVPFLND SKLE PDVRY PVLNYTYKKL GDKDNLPYKT RVKGLTTPWS PWNPFQV

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Macromolecule #3: RNA (33-MER)

MacromoleculeName: RNA (33-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 10.404171 KDa
SequenceString:
GACUUAAUGU CACGGUACCC AAUUUUCUGC CCC

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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: 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: 59.0 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: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 377252

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