Computational protocol: Structure of an octameric form of the minichromosome maintenance protein from the archaeon Pyrococcus abyssi

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Protocol publication

[…] PabMCM was studied both by negative staining and cryo electron microscopy (EM) and single particle analysis (SPA). The protein sample was added to glow-discharged grids, blotted and washed twice before staining using 1% uranyl acetate. Data were collected on a FEI F20 FEG microscope, equipped with a TemCam-F816 (8kx8k) CCD camera. Images were collected under low dose mode at nominal magnification of 50,000x, at a final sampling of 1.51 Å/pixel at the specimen level. Negatively stained single particle images were selected interactively using the e2boxer.py program from the EMAN2 single particle analysis package and extracted into boxes. Image processing was performed using the IMAGIC-5 and Eman packages. The dataset was re-sampled at 6.04 Å/pixel. ~45,000 images with homogeneous staining were band-pass filtered with a high pass cutoff of 110 Å and a low pass cutoff of 18 Å. The single particle images were analysed by Multivariate Statistical Analysis with IMAGIC-5. The dataset was subjected to successive rounds of alignment and classification in order to improve the resulting image class-averages. Three-dimensional reconstruction was performed using Imagic and Eman2 protocols. The map was deposited in the PDB with accession code EMD-3487. The cryoEM data collection was performed on Quantifoil grids covered with a thin layer of carbon, plunge-frozen into liquid ethane using a Vitrobot instrument. Data collection was performed at the same magnification of the negative staining study (50,000x), in low-dose mode. The single particle analysis was performed using the Relion software according to instructions. Figures were prepared with UCSF Chimera and Pymol. […]

Pipeline specifications

Software tools e2boxer.py, EMAN, IMAGIC, RELION, UCSF Chimera, PyMOL
Application cryo-EM
Organisms Pyrococcus abyssi
Diseases Sleep Initiation and Maintenance Disorders