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- EMDB-28072: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30... -

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Basic information

Entry
Database: EMDB / ID: EMD-28072
TitleCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Csx29
Map dataCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Csx29
Sample
  • Complex: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
KeywordsCRISPR / endonuclease / endopeptidase / RNA BINDING PROTEIN-RNA complex
Biological speciesDesulfonema ishimotonii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsDemircioglu FE / Wilkinson ME / Strecker J / Li D / Faure G / Macrae RK / Zhang F
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1DP1-HL141201 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2R01HG009761-05 United States
CitationJournal: Science / Year: 2022
Title: RNA-activated protein cleavage with a CRISPR-associated endopeptidase.
Authors: Jonathan Strecker / F Esra Demircioglu / David Li / Guilhem Faure / Max E Wilkinson / Jonathan S Gootenberg / Omar O Abudayyeh / Hiroshi Nishimasu / Rhiannon K Macrae / Feng Zhang /
Abstract: In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have ...In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.
History
DepositionSep 8, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28072.png

Downloads & links

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Map

FileDownload / File: emd_28072.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Csx29
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 320 pix.
= 318.24 Å		0.99 Å/pix.
x 320 pix.
= 318.24 Å		0.99 Å/pix.
x 320 pix.
= 318.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9945 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.047623932 - 0.104719736
Average (Standard dev.)0.00004226039 (±0.002518963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 318.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28072_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cas7-11 in complex with DR-mismatched target RNA, Csx29...

Fileemd_28072_half_map_1.map
AnnotationCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Csx29, half map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cas7-11 in complex with DR-mismatched target RNA, Csx29...

Fileemd_28072_half_map_2.map
AnnotationCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30, focus refined on Csx29, half map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30

EntireName: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
Components
  • Complex: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30

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Supramolecule #1: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30

SupramoleculeName: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Desulfonema ishimotonii (bacteria)

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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: 8
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: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 65382
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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