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- EMDB-27259: gRAMP-match PFS target -

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Basic information

Entry
Database: EMDB / ID: EMD-27259
TitlegRAMP-match PFS target
Map data
Sample
  • Complex: gRAMP-match PFS target
    • 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*UP*A)-3')
  • Ligand: ZINC ION
KeywordsCRISPR / GRAMP / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology: / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / RNA binding / RAMP superfamily protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 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 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27259.png

Downloads & links

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Map

FileDownload / File: emd_27259.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.28 Å/pix.
x 250 pix.
= 321. Å		1.28 Å/pix.
x 250 pix.
= 321. Å		1.28 Å/pix.
x 250 pix.
= 321. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.284 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0626
Minimum - Maximum-0.57066745 - 1.0549655
Average (Standard dev.)0.0010158252 (±0.020071939)
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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Half map: #2

Fileemd_27259_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_27259_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : gRAMP-match PFS target

EntireName: gRAMP-match PFS target
Components
  • Complex: gRAMP-match PFS target
    • 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*UP*A)-3')
  • Ligand: ZINC ION

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Supramolecule #1: gRAMP-match PFS target

SupramoleculeName: gRAMP-match PFS target / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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: 143.1015 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 LCRIEVIKKE VL SEDHNDE LRKQAEVIVE AFKQNDKLEK IRILADAIRT LRLHGEGVIE KDELPDGKEE RDKGHHLWDI KVQGTALRTK LKE LWQSNK DIGWRKFTEM LGSNLYLIYK KETGIETKEW IIVGRLKAAT PFYFGVQQPS DSIPGVINEH TSFNILLDKE NRYR IPRSA LRGALRRDLR TAFGSGCNVS LGGQILCNCK VCIEMRRITL KDSVSDFSEP PEIRYRIAKN PGTATVEDGS LFDIE VGPE GLTFPFVLRY RGHKFPEQLS SVIRYWEEND GKNGMAWLGG LDSTGKGRFA LKDIKIFEWD LNQKINEYIK ERGMRG KEK ELLEMGESSL PDGLIPYKFF EERECLFPYK ENLKPQWSEV QYTIEVGSPL LTADTISALT EPGNRDAIAY KKRVYND GN NAIEPEPRFA VKSETHRGIF RTAVGRRTGD LGKEDHEDCT CDMCIIFGNE HESSKIRFED LELINGNEFE KLEKHIDH V AIDRFTGGAL DKAKFDTYPL AGSPKKPLKL KGRFWIKKGF SGDHKLLITT ALSDIRDGLY PLGSKGGVGY GWVAGISID DNVPDDFKEM INKTEAAAAA AAAAAAAAAA AAAAKNKNIY YPHYFLDSGS KVYREKDIIT HEEFTEELLS GKINCKLETL TPLIIPDTS DENGLKLQGN KPGHKNYKFF NINGELMIPG SELRGMLRTH FEALTKSCFA IFGEDSTLSW RRKCASKTLG G KLDKALHP CTGLSDGLCP GCHLFGTTDY KGRVKFGFAK YENGPEWLIT RGNNPERSLT LGVLESPRPA FSIPDDESEI PG RKFYLHH NGWRIIRQKQ LEIRETVQPE RNVTTEVMDK GNVFSFDVKF ENLREWELGL LLQSLDPGKN IAHKLGKGKP YGF GSVKIK IDSLHTFKIN SNNDKIKRVP QSDIREYINK GYQKLIEWSG NNSIQKGNVL PQWHVIPHID KLYKLLWVPF LNDS KLEPD VRYPVLNEES KGYIEGSDYT YKKLGDKDNL PYKTRVKGLT TPWSPWNPFQ V

UniProtKB: RAMP superfamily protein, RAMP superfamily protein

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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*UP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 6.832134 KDa
SequenceString:
UCCGGGGCAG AAAAUUGGGU 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 TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 46269
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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