Type III-B CRISPR module RAMP protein Cmr1 / GGDEF domain-containing protein / Type III-B CRISPR module-associated protein Cmr3 / CRISPR type III-associated protein domain-containing protein / CRISPR system Cmr subunit Cmr5 / CRISPR type III-associated protein domain-containing protein Similarity search - Component
Journal: To Be Published Title: A new in vitro reconstitution system of type III-B CRISPR-Cas effector for functional and structural studies Authors: Ouchi R / Numata T
Name: ZINC ION / type: ligand / ID: 10 / Number of copies: 1 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.8 mg/mL
Buffer
pH: 7 Component:
Concentration
Name
20.0 mM
Tris-HCl
200.0 mM
NaCl
1.0 mM
DTT
2.0 mM
MgCl2
Grid
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.007 kPa
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
JEOL CRYO ARM 300
Specialist optics
Energy filter - Name: In-column Omega Filter
Image recording
Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Number grids imaged: 1 / Number real images: 5500 / Average exposure time: 2.597 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 80152
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)
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Atomic model buiding 1
Initial model
Chain - Source name: AlphaFold / Chain - Initial model type: in silico model Details: The initial model containing all protein subunits was constructed by AlphaFold2
Output model
PDB-9l7c: Type III-B CRISPR-Cas effector from Archaeoglobus fulgidus (Form 1)
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