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- EMDB-27252: gRAMP non-match PFS target RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-27252
TitlegRAMP non-match PFS target RNA
Map data
Sample
  • Complex: Gramp-Non matching PFS target
    • RNA: RNA (35-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*A)-3')
  • Protein or peptide: RAMP superfamily protein
  • Ligand: ZINC ION
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: 3.6 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 8, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27252.png

Downloads & links

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Map

FileDownload / File: emd_27252.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 Level1: 0.196
Minimum - Maximum-0.7509065 - 1.4607397
Average (Standard dev.)-0.0008683449 (±0.03423026)
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_27252_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27252_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_27252_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Gramp-Non matching PFS target

EntireName: Gramp-Non matching PFS target
Components
  • Complex: Gramp-Non matching PFS target
    • RNA: RNA (35-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*A)-3')
  • Protein or peptide: RAMP superfamily protein
  • Ligand: ZINC ION

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Supramolecule #1: Gramp-Non matching PFS target

SupramoleculeName: Gramp-Non matching PFS target / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #2-#3
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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: 197.219141 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 LCRIEVIKKS ES PLPSDTK EQSYTKDDTV EVLSEDHNDE LRKQAEVIVE AFKQNDKLEK IRILADAIRT LRLHGEGVIE KDELPDGKEE RDK GHHLWD IKVQGTALRT KLKELWQSNK DIGWRKFTEM LGSNLYLIYK KETGGVSTRF RILGDTEYYS KAHDSEGSDL FIPV TPPEG IETKEWIIVG RLKAATPFYF GVQQPSDSIP GKEKKSEDSL VINEHTSFNI LLDKENRYRI PRSALRGALR RDLRT AFGS GCNVSLGGQI LCNCKVCIEM RRITLKDSVS DFSEPPEIRY RIAKNPGTAT VEDGSLFDIE VGPEGLTFPF VLRYRG HKF PEQLSSVIRY WEENDGKNGM AWLGGLDSTG KGRFALKDIK IFEWDLNQKI NEYIKERGMR GKEKELLEMG ESSLPDG LI PYKFFEEREC LFPYKENLKP QWSEVQYTIE VGSPLLTADT ISALTEPGNR DAIAYKKRVY NDGNNAIEPE PRFAVKSE T HRGIFRTAVG RRTGDLGKED HEDCTCDMCI IFGNEHESSK IRFEDLELIN GNEFEKLEKH IDHVAIDRFT GGALDKAKF DTYPLAGSPK KPLKLKGRFW IKKGFSGDHK LLITTALSDI RDGLYPLGSK GGVGYGWVAG ISIDDNVPDD FKEMINKTEM PLPEEVEES NNGPINNDYV HPGHQSPKQD HKNKNIYYPH YFLDSGSKVY REKDIITHEE FTEELLSGKI NCKLETLTPL I IPDTSDEN GLKLQGNKPG HKNYKFFNIN GELMIPGSEL RGMLRTHFEA LTKSCFAIFG EDSTLSWRMN ADEKDYKIDS NS IRKMESQ RNPKYRIPDE LQKELRNSGN GLFNRLYTSE RRFWSDVSNK FENSIDYKRE ILRCAGRPKN YKGGIIRQRK DSL MAEELK VHRLPLYDNF DIPDSAYKAN DHCRKSATCS TSRGCRERFT CGIKVRDKNR VFLNAANNNR QYLNNIKKSN HDLY LQYLK GEKKIRFNSK VITGSERSPI DVIAELNERG RQTGFIKLSG LNNSNKSQGN TGTTFNSGWD RFELNILLDD LETRP SKSD YPRPRLLFTK DQYEYNITKR CERVFEIDKG NKTGYPVDDQ IKKNYEDILD SYDGIKDQEV AERFDTFTRG SKLKVG DLV YFHIDGDNKI DSLIPVRISR KCASKTLGGK LDKALHPCTG LSDGLCPGCH LFGTTDYKGR VKFGFAKYEN GPEWLIT RG NNPERSLTLG VLESPRPAFS IPDDESEIPG RKFYLHHNGW RIIRQKQLEI RETVQPERNV TTEVMDKGNV FSFDVKFE N LREWELGLLL QSLDPGKNIA HKLGKGKPYG FGSVKIKIDS LHTFKINSNN DKIKRVPQSD IREYINKGYQ KLIEWSGNN SIQKGNVLPQ WHVIPHIDKL YKLLWVPFLN DSKLEPDVRY PVLNEESKGY IEGSDYTYKK LGDKDNLPYK TRVKGLTTPW SPWNPFQVI AEHEEQEVNV TGSRPSVTDK IERDGKMV

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Macromolecule #2: RNA (35-MER)

MacromoleculeName: RNA (35-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 11.078582 KDa
SequenceString:
ACUUAAUGUC ACGGUACCCA AUUUUCUGCC CCGGA

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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*AP*CP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 6.815149 KDa
SequenceString:
UCCGGGGCAG AAAAUUGGAC A

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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 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

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: 89342

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