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- EMDB-27261: gRAMP-TPR-CHAT Non match PFS target RNA(Craspase) -

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Basic information

Entry
Database: EMDB / ID: EMD-27261
TitlegRAMP-TPR-CHAT Non match PFS target RNA(Craspase)
Map data
Sample
  • Complex: gRAMP-TPR-CHAT with Non match PFS target RNA Craspase
    • Protein or peptide: CHAT domain protein
    • 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*AP*CP*GP*AP*U)-3')
  • 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: 2.57 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_27261.png

Downloads & links

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Map

FileDownload / File: emd_27261.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: 0.14
Minimum - Maximum-1.1555538 - 2.602391
Average (Standard dev.)-0.00026588608 (±0.055240836)
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_27261_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27261_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_27261_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 Non match PFS target RNA Craspase

EntireName: gRAMP-TPR-CHAT with Non match PFS target RNA Craspase
Components
  • Complex: gRAMP-TPR-CHAT with Non match PFS target RNA Craspase
    • Protein or peptide: CHAT domain protein
    • 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*AP*CP*GP*AP*U)-3')
  • Ligand: ZINC ION

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

SupramoleculeName: gRAMP-TPR-CHAT with Non 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: 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: 78.403711 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: PTREDIDRKE AERLLDEAFN PRTKPVDRKK IINSALKILI GLYKEKKDDL TSASFISIAR AYYLVSITIL PKGTTIPEKK KEALRKGIE FIDRAINKFN GSILDSQRAF RIKSVLSIEF NRIDREKCDN IKLKNLLNEA VDKGCTDFDT YEWDIQIAIR L CELGVDME ...String:
PTREDIDRKE AERLLDEAFN PRTKPVDRKK IINSALKILI GLYKEKKDDL TSASFISIAR AYYLVSITIL PKGTTIPEKK KEALRKGIE FIDRAINKFN GSILDSQRAF RIKSVLSIEF NRIDREKCDN IKLKNLLNEA VDKGCTDFDT YEWDIQIAIR L CELGVDME GHFDNLIKSN KANDLQKAKA YYFIKKDDHK AKEHMDKCTA SLKYTPCSHR LWDETVGFIE RLKGDSSTLW RD FAIKTYR SCRVQEKETG TLRLRWYWSR HRVLYDMAFL AVKEQADDEE PDVNVKQAKI KKLAEISDSL KSRFSLRLSD MEK MPKSDD ESNHEFKKFL DKCVTAYRSI YVINRKLLEL TQVPEGWVVV HFYLNKLEGM GNAIVFDKCA NSWQYKEFQY KELF EVFLT WQANYNLYKE NAAEHLVTLC KKIGETMPFL FCDNFIPNGK DVLFVPHDFL HRLPLHGSIE NKTNGKLFLE NHSCC YLPA WSFASEKEAS TSDEYVLLKN FDQGHFETLQ NNQIWGTQSV KDGASSDDLE NIRNNPRLLT ILCHGEANMS NPFRSM LKL ANGGITYLEI LNSVKGLKGS QVILGACETD LVPPLSDVMD EHYSVATALL LIGAAGVVGT MWKVRSNKTK SLIEWKL EN IEYKLNEWQK ETGGAAYKDH PPTFYRSIAF RSIGFPL

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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: 147.151625 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 STRFRILGDT EYYSKAHDSE GSDLFIPVTP PEGIETKEWI IVGRLKAATP FYFG VQQPS DSIPGKEKKS EDSLVINEHT SFNILLDKEN RYRIPRSALR GALRRDLRTA FGSGCNVSLG GQILCNCKVC IEMRR ITLK DSVSDFSEPP EIRYRIAKNP GTATVEDGSL FDIEVGPEGL TFPFVLRYRG HKFPEQLSSV IRYWEENDGK NGMAWL GGL DSTGKGRFAL KDIKIFEWDL NQKINEYIKE RGMRGKEKEL LEMGESSLPD GLIPYKFFEE RECLFPYKEN LKPQWSE VQ YTIEVGSPLL TADTISALTE PGNRDAIAYK KRVYNDGNNA IEPEPRFAVK SETHRGIFRT AVGRRTGDLG KEDHEDCT C DMCIIFGNEH ESSKIRFEDL ELINGNEFEK LEKHIDHVAI DRFTGGALDK AKFDTYPLAG SPKKPLKLKG RFWIKKGFS GDHKLLITTA LSDIRDGLYP LGSKGGVGYG WVAGISIDDN VPDDFKEMIN KTNNDYVHPG HQSPKQDHKN KNIYYPHYFL DSGSKVYRE KDIITHEEFT EELLSGKINC KLETLTPLII PDTSDENGLK LQGNKPGHKN YKFFNINGEL MIPGSELRGM L RTHFEALT KSCFAIFGED STLSWASKTL GGKLDKALHP CTGLSDGLCP GCHLFGTTDY KGRVKFGFAK YENGPEWLIT RG NNPERSL TLGVLESPRP AFSIPDDESE IPGRKFYLHH NGWRIIRQKQ LEIRETVQPE RNVTTEVMDK GNVFSFDVRF ENL REWELG LLLQSLDPGK NIAHKLGKGK PYGFGSVKIK IDSLHTFKIN SNNDKIKRVP QSDIREYINK GYQKLIEWSG NNSI QKGNV LPQWHVIPHI DKLYKLLWVP FLNDSKLEPD VRYPVLNEES KGYIEGSDYT YKKLGDKDNL PYKTRVKGLT TPWSP WNPF QV

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Macromolecule #3: RNA (36-MER)

MacromoleculeName: RNA (36-MER) / type: rna / ID: 3 / 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 #4: 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*AP*CP*GP*AP*U)-3')
type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 7.466521 KDa
SequenceString:
UCCGGGGCAG AAAAUUGGAC GAU

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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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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: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 340526

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