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- EMDB-40297: CRISPR-Cas type III-D effector complex bound to a target RNA loca... -

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Basic information

Entry
Database: EMDB / ID: EMD-40297
TitleCRISPR-Cas type III-D effector complex bound to a target RNA local refinement map
Map dataCRISPR-Cas type III-D complex bound to target RNA local refinement map
Sample
  • Complex: CRISPR-Cas type III-D effector complex bound to target RNA
KeywordsCRISPR / CRISPR-Cas / type III / complex / RNA / crRNA / RNA BINDING PROTEIN-RNA complex
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsSchwartz EA / Taylor DW
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nat Commun / Year: 2024
Title: RNA targeting and cleavage by the type III-Dv CRISPR effector complex.
Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D ...Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D Fagerlund / David W Taylor /
Abstract: CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular ...CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.
History
DepositionApr 2, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40297.png

Downloads & links

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Map

FileDownload / File: emd_40297.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCRISPR-Cas type III-D complex bound to target RNA local refinement map
Voxel sizeX=Y=Z: 0.81 Å
Density
Contour LevelBy AUTHOR: 0.624
Minimum - Maximum-2.3844328 - 5.4240575
Average (Standard dev.)0.00064681727 (±0.06494863)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 356.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_40297_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_40297_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CRISPR-Cas type III-D effector complex bound to target RNA

EntireName: CRISPR-Cas type III-D effector complex bound to target RNA
Components
  • Complex: CRISPR-Cas type III-D effector complex bound to target RNA

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Supramolecule #1: CRISPR-Cas type III-D effector complex bound to target RNA

SupramoleculeName: CRISPR-Cas type III-D effector complex bound to target RNA
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Molecular weightTheoretical: 351 KDa

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHEPES-NaOH
100.0 mMPotassium ChlorideKCl
5.0 %Glycerol
1.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsParticles were monodisperse and homogeneous.

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 609722
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model

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