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- EMDB-27257: Apo gRAMP -

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Basic information

Entry
Database: EMDB / ID: EMD-27257
TitleApo gRAMP
Map data
Sample
  • Complex: APO gRAMP
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (42-MER)
  • Ligand: ZINC ION
KeywordsCRISPR / GRAMP / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyCRISPR type III-associated protein / RAMP superfamily / defense response to virus / RAMP superfamily protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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_27257.png

Downloads & links

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Map

FileDownload / File: emd_27257.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.284 Å
Density
Contour LevelBy AUTHOR: 1.05
Minimum - Maximum-24.653358000000001 - 52.716859999999997
Average (Standard dev.)0.031320978 (±1.1281972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 321.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_27257_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27257_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27257_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 : APO gRAMP

EntireName: APO gRAMP
Components
  • Complex: APO gRAMP
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (42-MER)
  • Ligand: ZINC ION

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Supramolecule #1: APO gRAMP

SupramoleculeName: APO gRAMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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: 184.351406 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 FGETLSWRMN ADEKDYKIDS NSIRKMESQR NPKYRIPDEL QKELRNSGNG LFNRLYTSER RFWSDVSNKF EN SIDYKRE ILRCAGRPKN YKGGIIRQRK DSLMAEELKV HRLPLYDNFD IPDSAYKAND HCRKSATCST SRGCRERFTC GIK VRDKNR VFLNAANNNR QYLNNIKKSN HDLYLQYLKG EKKIRFNSKV ITGSERSPID VIAELNERGR QTGFIKLSGL NNSN KSQGN TGTTFNSGWD RFELNILLDD LETRPSKSDY PRPRLLFTKD QYEYNITKRC ERVFEIDKGN KTGYPVDDQI KKNYE DILD SYDGIKDQEV AERFDTFTRG SKLKVGDLVY FHIDGDNKID SLIPVRGKLD KALHPCTGLS DGLCPGCHLF GTTDYK GRV KFGFAKYENG PEWLITRGNN PERSLTLGVL ESPRPAFSIP DDESEIPGRK FYLHHNGWRI IRQKQLEIRE TVQPERN VT TEVMDKGNVF SFDVRFENLR EWELGLLLQS LDPGKNIAHK LGKGKPYGFG SVKIKIDSLH TFKIIKRVPQ SDIREYIN K GYQKLIEWSL PQWHVIPHID KLYKLLWVPF LNDSKLEPDV RYPVLNYTYK KLGDKDNLPY KTRVKGLTTP WSPWNPFQV

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Macromolecule #2: RNA (42-MER)

MacromoleculeName: RNA (42-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 13.295887 KDa
SequenceString:
UUAAUGUCAC GGUACCCAAU UUUCUGCCCC GGACUCCACG GC

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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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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 TALOS ARCTICA
Electron beamAcceleration voltage: 200 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: Talos Arctica / 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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 21741

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