Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

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Three-Dimensional Reconstruction of Neuronal Mitochondria by Electron Tomography

Neuromethods - Techniques to Investigate Mitochondrial Function in Neurons ◽

10.1007/978-1-4939-6890-9_1 ◽

2017 ◽

pp. 1-29

Author(s):

Guy Perkins

Keyword(s):

Electron Tomography ◽

Three Dimensional ◽

Three Dimensional Reconstruction ◽

Dimensional Reconstruction

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Challenges of three-dimensional reconstruction of ribonucleoprotein complexes from electron spectroscopic images - Reconstructing ribosomal RNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162727 ◽

1996 ◽

Vol 54 ◽

pp. 52-53

Author(s):

D.R. Beniac ◽

G.J. Czarnota ◽

T.A. Bartlett ◽

F.P. Ottensmeyer ◽

G. Harauz

Keyword(s):

Ribosomal Rna ◽

Correlation Functions ◽

Three Dimensional ◽

Three Dimensional Reconstruction ◽

Mutual Correlation ◽

Ribosomal Subunits ◽

E Coli ◽

Ribonucleoprotein Complexes ◽

Dimensional Reconstruction

Transmission electron microscopy has been dominant in structural studies of the ribosome and its constituent ribonucleic acids and proteins. Ribosomal RNA (rRNA) has central importance in the architecture of this complex and in protein synthesis. Our work has entailed using electron spectroscopic imaging (ESI) to probe the tertiary structure of rRNA in situ in a prokaryote (Escherichia coli) and in a eukaryote (Thermomyces lanuginosus). ESI uses only electrons which have lost a specific amount of energy due to specific inner-shell ionisation interactions with the specimen to form an elemental map. In nucleoprotein complexes, a map of the phosphorus distribution represents primarily a projection of the phosphate backbone of the nucleic acid component. The visualisation of rRNA in situ in the intact ribosomal subunit by ESI was demonstrated almost a decade ago to be feasible. Our work on quantitative image analysis of ES images of E. coli and Th. lanuginosus ribosomal subunits has presented unique challenges and has resulted in new algorithmic developments generally applicable to such images. These innovations include a singular pretreatment procedure, the use of mutual correlation functions rather than cross correlation functions to reduce the effect of low spatial frequency components, and angular determination using iterative quaternion-assisted angular reconstitution to compute a three-dimensional reconstruction. These investigations have produced direct information regarding ribosomal rRNA localisation in the ribosomal subunits of E. coli and Th. lanuginosus, and the position of non-conserved sequences.

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