Architecture and self-assembly of the jumbo bacteriophage nuclear shell - Nature

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Architecture and self-assembly of the jumbo bacteriophage nuclear shell - Nature
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Nature research paper: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

Protein expression and purification

Full-length Chimallin from bacteriophages 201phi2-1 and Goslar were cloned with an N-terminal TEV protease-cleavable Histag using UC Berkeley Macrolab vector 2-BT . Truncations and other modified constructs were cloned by PCR mutagenesis and isothermal assembly, and inserted into the same vector. Proteins were expressed in= 0.8, inducing expression with 0.3 mM IPTG, then growing cells at 20 °C for 16–18 h.

All samples were imaged using a Titan Krios G3 transmission electron microscope operated at 300 kV configured for fringe-free illumination and equipped with a K2-directed electron detector mounted post Quantum 968 LS imaging filter . The microscope was operated in EFTEM mode with a slit-width of 20 eV and using a 70 µm objective aperture. Automated data acquisition was performed using SerialEM-v3.8b11For the 201phi2-1 24mer sample, tilt series were acquired using a pixel size of 1.

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