A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor.
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 approximately 660-kDa ATP-fueled AAA+ motor to 7.5 A 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.
Elmlund
Hans
H
Department of Biosciences and Nutrition, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, Novum, SE-141 87 Huddinge, Sweden.
Lundqvist
Joakim
J
Al-Karadaghi
Salam
S
Hansson
Mats
M
Hebert
Hans
H
Lindahl
Martin
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000465:Algorithms; D000595:Amino Acid Sequence; D003198:Computer Simulation; D020285:Cryoelectron Microscopy / Q000379:methods*; D019281:Dimerization; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D005583:Fourier Analysis; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D015203:Reproducibility of Results; D016354:Rhodobacter capsulatus / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013816:Thermodynamics
D021122:Protein Subunits; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004398:Dyneins; D008190:Lyases; C086788:magnesium chelatase