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- EMDB-34158: Structure of the SbCas7-11-crRNA-NTR complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34158
TitleStructure of the SbCas7-11-crRNA-NTR complex
Map data
Sample
  • Complex: ternary complex
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (32-MER)
    • RNA: RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
  • Ligand: ZINC ION
KeywordsComplex / RNA-binding / RNA BINDING PROTEIN
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: 2.8 Å
AuthorsYu G / Wang X / Deng Z / Zhang H
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32071218 China
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Target RNA-guided protease activity in type III-E CRISPR-Cas system.
Authors: Xiaoshen Wang / Guimei Yu / Yanan Wen / Qiyin An / Xuzichao Li / Fumeng Liao / Chengwei Lian / Kai Zhang / Hang Yin / Yong Wei / Zengqin Deng / Heng Zhang /
Abstract: The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, ...The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.
History
DepositionAug 23, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34158.png

Downloads & links

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Map

FileDownload / File: emd_34158.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.5
Minimum - Maximum-37.297935000000003 - 56.937427999999997
Average (Standard dev.)0.000000000003875 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 285.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_34158_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_34158_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 : ternary complex

EntireName: ternary complex
Components
  • Complex: ternary complex
    • Protein or peptide: RAMP superfamily protein
    • RNA: RNA (32-MER)
    • RNA: RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
  • Ligand: ZINC ION

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Supramolecule #1: ternary complex

SupramoleculeName: ternary complex / 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: 197.173125 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (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 VINEHASFNI 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 AAIAYKKRVY 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 FSFDVRFE N LREWELGLLL QSLDPGKNIA HKLGKGKPYG FGSVKIKIDS LHTFKINSNN DKIKRVPQSD IREYINKGYQ KLIEWSGNN SIQKGNVLPQ WHVIPHIDKL YKLLWVPFLN DSKLEPDVRY PVLNEESKGY IEGSDYTYKK LGDKDNLPYK TRVKGLTTPW SPWNPFQVI AEHEEQEVNV TGSRPSVTDK IERDGKMV

UniProtKB: RAMP superfamily protein

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Macromolecule #2: RNA (32-MER)

MacromoleculeName: RNA (32-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 10.058966 KDa
SequenceString:
ACUUAAUGUC ACGGUACCCA AUUUUCUGCC CC

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Macromolecule #3: RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')

MacromoleculeName: RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Molecular weightTheoretical: 14.915912 KDa
SequenceString:
CUCUAGUAAC AGCCGUGGAG UCCGGGGCAG AAAAUUGGGU GAUUAA

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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

Concentration0.6 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsNo further treatment.

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 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: 0.5 µm

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Image processing

Particle selectionNumber selected: 2016303
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 442994
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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