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- EMDB-15820: Structure of the type I-G CRISPR effector -

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Basic information

Entry
Database: EMDB / ID: EMD-15820
TitleStructure of the type I-G CRISPR effector
Map dataMap from the Csx17 Multibody segment
Sample
  • Complex: Type I-G cascade complex large subunit CSX17 protein
    • Protein or peptide: Type I-G CRISPR Cascade large subunit CSX17
Function / homologyCRISPR-associated protein Csx17, subtype Dpsyc / Type I-U CRISPR-associated protein Csx17
Function and homology information
Biological speciesThioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsShangguan Q / Graham S / Sundaramoorthy R / White MF
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S000313/1 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structure and mechanism of the type I-G CRISPR effector.
Authors: Qilin Shangguan / Shirley Graham / Ramasubramanian Sundaramoorthy / Malcolm F White /
Abstract: Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 ...Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.
History
DepositionSep 15, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15820.png

Downloads & links

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Map

FileDownload / File: emd_15820.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap from the Csx17 Multibody segment
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 350 pix.
= 352.8 Å		1.01 Å/pix.
x 350 pix.
= 352.8 Å		1.01 Å/pix.
x 350 pix.
= 352.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.008 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0044
Minimum - Maximum-0.027057964 - 0.05444887
Average (Standard dev.)2.6254973e-05 (±0.00061734015)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 352.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map from the Csx17 Multibody segment

Fileemd_15820_half_map_1.map
AnnotationHalf Map from the Csx17 Multibody segment
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map from the Csx17 Multibody segment

Fileemd_15820_half_map_2.map
AnnotationHalf Map from the Csx17 Multibody segment
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Type I-G cascade complex large subunit CSX17 protein

EntireName: Type I-G cascade complex large subunit CSX17 protein
Components
  • Complex: Type I-G cascade complex large subunit CSX17 protein
    • Protein or peptide: Type I-G CRISPR Cascade large subunit CSX17

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Supramolecule #1: Type I-G cascade complex large subunit CSX17 protein

SupramoleculeName: Type I-G cascade complex large subunit CSX17 protein / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 7.926 KDa

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Macromolecule #1: Type I-G CRISPR Cascade large subunit CSX17

MacromoleculeName: Type I-G CRISPR Cascade large subunit CSX17 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thioalkalivibrio sulfidiphilus HL-EbGr7 (bacteria)
Strain: HL-EbGR7
Molecular weightTheoretical: 79.372023 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDKDMHINEI VLRGCAPTPL AAYLKALGVL RLVCEQVDAT AKGWWQDECF MLRTRLDDND LRRFFIEDYR PTPMLSPWNG GSGFYRKGN ETAWSTLEKI ITTQAERWRP FRDTAEVMAD ALEHLKLTEK PAELDKRALL ARLRATLDDE FLPWLDAAVL L TDDKPDYP ...String:
MDKDMHINEI VLRGCAPTPL AAYLKALGVL RLVCEQVDAT AKGWWQDECF MLRTRLDDND LRRFFIEDYR PTPMLSPWNG GSGFYRKGN ETAWSTLEKI ITTQAERWRP FRDTAEVMAD ALEHLKLTEK PAELDKRALL ARLRATLDDE FLPWLDAAVL L TDDKPDYP PLLGTGGNDG RLDFTSNYMQ RLLEMFDPVT GKAQGDVGNK LESALFARPV PGMTALAIGQ FSPGAAGGPN SS TGFDSGA QVNIWDYVLM LEGALLFAAT ATRRLESADP SALSYPFTVR PSGGGSGAVA LGDERPARAE IWMPLWERPA SLP ELRVLL GEGRVTLNGR LPRDGLDFAR AVAKLGTDRG VRAFQRYAFM MRSGKAYLAT PLNRFHVHRN PKADLIDQLE RGDW LRRFR RAARSTHAPA RLQGLAHRLD DALFDLVRVA DPRRVQEVLK VLGEVQFYLA LSPSLREQVR PVPRLDAHWV EAARD DSHE FRVAAALAGL DDGLPMGVHL APIDPVKRNV WAPESRLAVW GQGNLSDNLA QVLQRRLLTA SRTDLNDKPL SGRCPA DEG AVAAFLAGDA DERRIAELMA GLACARLPAR LPLRQRGASE ASSLPMIYAL LKPLFVPDAQ LREAAVLTPD GCLPLPP AL PRLLRAGPAG VGRAVDLARR RRRASGLADA GWRLTPPYPD GGRLLAALMI PVEIRVIKGF IKRLADHKSD EPATQDAS

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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

Concentration1 mg/mL
BufferpH: 7.5 / Details: 20mM Tris-HCl, 250mM NaCl, pH 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV / Details: blot time 3.5, blot force 4.
DetailsSize exclusion purified monodisperse sample in solution

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.8000000000000003 µm / Calibrated defocus min: 1.6 µm / Calibrated magnification: 105000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 183.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Sampling interval: 0.84 µm / Number grids imaged: 1 / Number real images: 15710 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1000
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: INSILICO MODEL / In silico model: RELION INITIAL MODEL / Details: Using stochastic gradient method
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4)
Final 3D classificationNumber classes: 2 / Avg.num./class: 2300000 / Software - Name: RELION (ver. 4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Details: Multi-body refinement. / Number images used: 415000

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 188 / Target criteria: Correlation Coefficient
Output model

PDB-8b2x:
Structure of the type I-G CRISPR effector

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