Similar protocols

Pipeline publication

[…] microscopy (EM) with a buffer containing 25 mM Tris (pH 7.4) and 250 mM NaCl. An aliquot of 2.5 µl of each of the diluted samples was placed onto a carbon film-coated, glow-discharged 300-mesh copper grid. Excess sample was blotted away by filter paper after one minute. The sample was immediately stained with 2% uranyl acetate solution and air dried. Electron micrographs were recorded in an FEI TF20 electron microscope (operated at 200 kV) on a TVIPS 16-megapixel CCD camera at 70,000× magnification. The sample with optimal particle concentration as shown in was used for extended imaging and in-depth 3D reconstruction analysis., First, we picked 500 particles manually from 5 CCD images with EMAN boxer program and obtained a preliminary reconstruction to 22 Å resolution with EMAN . Approximately 20,000 particles were subsequently picked automatically from 30 CCD images with SIGNATURE , using the previous 22 Å structure as reference. D3 symmetry was imposed during refinement and 3D reconstruction. Approximately 12,000 particles were selected for the final 3D reconstruction. The resolution of the final 3D reconstruction was assessed to be 12 Å based on the 0.5 cutoff in the Fourier shell correlation between 3D reconstructions from the two half sets of the whole data set (). The UCSF Chimera program was used to create 3D graphical representations . The atomic model of MCC was fitted to the density map using the fit-model-to-map module of Chimera., Site-specific mutations were introduced with the QuikChange kit (Agilent) and sequenced for confirmation. The mutant plasmids were transformed into E. coli, and the formation of the PaMCC holoenzyme was assessed by nickel affinity chromatography., The catalytic activity of PaMCC was determined using a coupled enzyme assay, converting the hydrolysis of ATP to the disappearance of NADH ,. The reaction mixture contained 100 mM HEPES (pH 8.0), 0.5 mM ATP, 8 mM MgCl2, 40 mM KHCO3, 0.5 mM methylcrotonyl-CoA or crotonyl-CoA, 0.2 […]

Pipeline specifications

Software tools EMAN, FSC, UCSF Chimera