Journal: Nucleic Acids Res / Year: 2016 Title: 3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM. Authors: Eugene Y D Chua / Vinod K Vogirala / Oviya Inian / Andrew S W Wong / Lars Nordenskiöld / Juergen M Plitzko / Radostin Danev / Sara Sandin / Abstract: The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining ...The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein-DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.
Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Details: 3-4 seconds blotting time.
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Imaging
Microscope: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 31 e/Å2 / Illumination mode: SPOT SCAN
Lens
Imaging mode: BRIGHT FIELD
Specimen Holder
Model: OTHER
Camera
Detector: GATAN K2 SUMMIT (4k x 4k)
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Image processing
Processing
Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 26060 Details: The recorded micrographs were clipped to remove dust and the movie frames were motion-corrected.
3D reconstruction
Algorithm: FOURIER SPACE / Software: RELION / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Details: RELION 1.4 was used for the final reconstruction.
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