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A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor |
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| Authors: | Elmlund Hans Lundqvist Joakim Al-Karadaghi Salam Hansson Mats Hebert Hans Lindahl Martin |
| Affiliation: | 1 Department of Biosciences and Nutrition, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, Novum, SE-141 87 Huddinge, Sweden 2 Department of Molecular Biophysics, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden 3 Department of Biochemistry, Lund University, SE-221 00, Lund, Sweden |
| Abstract: | The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an ∼ 660-kDa ATP-fueled AAA+ motor to 7.5 Å resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice. |
| Keywords: | 3D, three-dimensional 2D, two-dimensional cryo-EM, cryo-electron microscopy PDB, Protein Data Bank SA, simulated annealing PCA, principal component analysis FT, Fourier transform CTF, contrast transfer function FSC, Fourier shell correlation HslU, heat shock locus U SNR, signal-to-noise ratio |
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