title: Protein secondary structure determination by constrained single-particle cryo-electron tomography.
abstract: Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be achieved with single-particle cryo-EM are frequently limited by inaccuracies in assigning molecular orientations based solely on 2D projection images. Tomographic data collection schemes, however, provide powerful constraints that can be used to more accurately determine molecular orientations necessary for 3D reconstruction. Here, we propose "constrained single-particle tomography" as a general strategy for 3D structure determination in cryo-EM. A key component of our approach is the effective use of images recorded in tilt series to extract high-resolution information and correct for the contrast transfer function. By incorporating geometric constraints into the refinement to improve orientational accuracy of images, we reduce model bias and overrefinement artifacts and demonstrate that protein structures can be determined at resolutions of ∼8 Å starting from low-dose tomographic tilt series.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Alberto
authors.initials: A
authors.lastname: Bartesaghi
authors.forename: Federico
authors.initials: F
authors.lastname: Lecumberry
authors.forename: Guillermo
authors.initials: G
authors.lastname: Sapiro
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
mesh_terms: D000465:Algorithms; D018834:Chaperonin 60 / Q000737:chemistry / Q000648:ultrastructure; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000648:ultrastructure; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000648:ultrastructure; D035581:Transcription Factor TFIIB / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C073680:SUA7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035581:Transcription Factor TFIIB; D012319:RNA Polymerase II