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Direct visualization of secondary structures of F-actin by electron cryomicroscopy

Nature volume 467, pages 724–728 (2010)Cite this article

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Abstract

F-actin is a helical assembly of actin, which is a component of muscle fibres essential for contraction and has a crucial role in numerous cellular processes, such as the formation of lamellipodia and filopodia1,2, as the most abundant component and regulator of cytoskeletons by dynamic assembly and disassembly (from G-actin to F-actin and vice versa). Actin is a ubiquitous protein and is involved in important biological functions, but the definitive high-resolution structure of F-actin remains unknown. Although a recent atomic model well reproduced X-ray fibre diffraction intensity data from a highly oriented liquid-crystalline sol specimen3, its refinement without experimental phase information has certain limitations. Direct visualization of the structure by electron cryomicroscopy, however, has been difficult because it is relatively thin and flexible. Here we report the F-actin structure at 6.6 Å resolution, made obtainable by recent advances in electron cryomicroscopy. The density map clearly resolves all the secondary structures of G-actin, such as α-helices, β-structures and loops, and makes unambiguous modelling and refinement possible. Complex domain motions that open the nucleotide-binding pocket on F-actin formation, specific D-loop and terminal conformations, and relatively tight axial but markedly loose interprotofilament interactions hydrophilic in nature are revealed in the F-actin model, and all seem to be important for dynamic functions of actin.

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Figure 1: High-contrast cryoEM image of F-actin, its power spectrum and angular distribution of rotation per subunit.
Figure 2: Three-dimensional density map of F-actin with a fitted atomic model in stereo.
Figure 3: Comparison of actin models in F-actin with the uncomplexed crystal structure 16
Figure 4: Axial and lateral interactions of actin subunits in F-actin.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The reconstructed density map has been deposited in the Electron Microscopy Data Bank under accession code EMD-5168, and the atomic coordinates have been deposited in the Protein Data Bank under accession code 3MFP.

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Acknowledgements

We thank T. Kato for setting up and managing our cryoEM and computing facilities for high-throughput, high-resolution image analysis, and I. Tokita, M. Urabe and JEOL for maintaining the electron cryomicroscope in the best conditions. We also thank E. Egelman for his help in the use of the iterative helical real-space refinement method in the early stages of this study, and F. Oosawa, S. Asakura, H. Hotani and D. L. D. Caspar for their discussions, continuous support and encouragement. T.F. was a research fellow of the Japan Society for the Promotion of Science. This work was supported in part by Grants-in-Aid for Scientific Research to K.N. (16087207 and 21227006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan ,

    Takashi Fujii, Atsuko H. Iwane, Toshio Yanagida & Keiichi Namba

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  1. Takashi Fujii
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  2. Atsuko H. Iwane
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  3. Toshio Yanagida
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Contributions

T.F. made various improvements to the cryoEM method and performed all the cryoEM experiments, the image analysis and the model-building of F-actin. A.H.I. prepared G-actin. T.Y. and K.N. planned the project, and K.N. supervised the project. T.F. and K.N. wrote the paper on the basis of discussions with A.H.I. and T.Y.

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Correspondence to Keiichi Namba.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-7 with legends, Supplementary Tables 1-2, legends for Supplementary Movies 1-2 and additional references. (PDF 15001 kb)

Supplementary Movie 1

This movie shows a side view of the F-actin density map at 6.6 Å resolution obtained by cryoEM image analysis - see Supplementary Information file page 11 for full legend. (MOV 8871 kb)

Supplementary Movie 2

In this move we see the initial model fitting to the cryoEM density map and its refinement process to the final F-actin model - see Supplementary Information file page 11 for full legend. (MOV 4220 kb)

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Fujii, T., Iwane, A., Yanagida, T. et al. Direct visualization of secondary structures of F-actin by electron cryomicroscopy. Nature 467, 724–728 (2010). https://doi.org/10.1038/nature09372

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