National Center for Macromolecular Imaging
Acrosomal Bundle
Michael B. Sherman, Michael Schmid, and Joanita Jakana in collaboration with Paul Matsudaira at MIT


Actin seldom exists in cells in a simple G-/F-actin equilibrium. Many binding proteins modulate polymerization, regulate the length of polymers or promote association into higher order assemblies, such as bundles. These bundles are sometimes found in extensions of the cell surface. Examples are filopodia, microvilli, and the acrosomal process. The function of a bundle of actin seems to be to stiffen actin beyond the normal rigidity of an F-actin filament. The biological outcome is a structure that is able to maintain and change the shape and size of various cells or cell organelles. Since actin alone does not usually form bundles and bundles usually contain other proteins, we should expect the cross-linking function to reside in these proteins.

One such bundle is the acrosomal process, an intracellular quasi-crystalline organelle in the head of the sperm of various invertebrates, including the horseshoe crab Limulus polyphemus. It extends from the anterior tip of the sperm through the nucleus, and coils around the base of the sperm head. When the sperm encounters an egg, calcium enters the sperm cell and causes the acrosomal bundle to straighten out and extend from the sperm head. It can be up to 60 Ám long, but has a diameter of only 0.1 Ám. In Limulus the bundle consists of about 100 actin filaments packed together in a pseudo-hexagonal lattice. There are two major proteins in a 1:1 stoichiometric ratio, actin (42 kDa) and scruin (102 kDa). Scruin-scruin interactions are believed to be responsible for cross-linking the actin filaments together to form the bundle.

In collaborating with Paul Matsudaira at MIT, we have targeted to determine the 3-dimensional structure of the acrosomal process beyond 10 ┼ resolution by the 400 keV electron cryomicroscopy. Because of its structural complexity, we have approached the structural analysis in a step-wise manner and have developed various computational means to understand the molecular interactions within each filament and between adjacent filaments.


Selected Images


Click on image for larger version

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2 and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits. Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2 and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.
The acrosomal bundle from Limulus sperm has been imaged to 7 ┼ in a 400kV electron cryomicroscope using the spot-scan technique. These images have been processed by Fourier averaging, corrected for the contrast transfer function, and merged to that resolution. The reconstructed density map of this projection shows features consistent with the 13 ┼ projection map from the helical reconstruction and with the x-ray derived model for F-actin, and regions of possible scruin-scruin contacts.  
The 7 ┼ projection mass density map of three unit cells of acrosomal bundle is seen in h01 view(a=147 ┼ and c=762 ┼). The light color represents the protein (high) density and the dark color represents the solvent (low) density. Arrow in the figure points to regions of scuin density. Outlined by a dotted line is the position of the best cross-correlation of the projection map from the 3D structure with the central unit cell. The acrosomal bundle from Limulus sperm has been imaged to 7 ┼ in a 400kV electron cryomicroscope using the spot-scan technique. These images have been processed by Fourier averaging, corrected for the contrast transfer function, and merged to that resolution. The reconstructed density map of this projection shows features consistent with the 13 ┼ projection map from the helical reconstruction and with the x-ray derived model for F-actin, and regions of possible scruin-scruin contacts.
The 7 ┼ projection mass density map of three unit cells of acrosomal bundle is seen in h01 view(a=147 ┼ and c=762 ┼). The light color represents the protein (high) density and the dark color represents the solvent (low) density. Arrow in the figure points to regions of scuin density. Outlined by a dotted line is the position of the best cross-correlation of the projection map from the 3D structure with the central unit cell.

Selected Publications

 
  • Schmid, M. F., Matsudaira, P., Jeng, T. W., Jakana, J., Towns-Andrews, E., Bordas, J. & Chiu, W. (1991). Crystallographic analysis of acrosomal bundle from Limulus sperm. J. Mol. Biol. 221, 711-725.
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  • Schmid, M. F., Jakana, J., Matsudaira, P. & Chiu, W. (1992). Effects of radiation damage with 400kV electrons on frozen, hydrated actin bundles. J. Struct. Biol. 108, 62-68.
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  • Schmid, M. F., Jakana, J., Matsudaira, P. & Chiu, W. (1993). Imaging frozen, hydrated acrosomal bundle from Limulus sperm at 7 ┼ resolution with a 400 kV electron cryomicroscope. J. Mol. Biol. 230, 384-386.
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  • Schmid, M. F., Agris, J., Jakana, J., Matsudaira, P. & Chiu, W. (1994). Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin. J. Cell Biol. 124, 341-350.
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  • Schmid, M. F., Jakana, J., Chiu, W. & Matsudaira, P. (1995). A 7 ┼ projection map of frozen, hydrated acrosomal bundle from Limulus sperm. J. Struct. Biol. 115, 209-213.
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  • Sherman, M. B., Jakana, J., Sun, S., Matsudaira, P., Chiu, W. & Schmid, M. F. (1997). A strategy for electron tomographic data collection and crystallographic reconstruction of biological bundles. J. Struct. Biol. 120, 245-256.
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  • Sherman, M.B., Jakana, J. Sun, S., Matsudaira, P., Chiu, W. and Schmid, M.F. (1999) The three-dimensional structure of the Limulus acrosomal process: A dynamic actin bundle. J. Mol. Biol. 294:139-149.
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  • Schmid, M.F. (2003). Cross-correlation and merging of crystallographic reflections derived from cryoelectron micrographs of 3D crystals: application to the Limulus acrosomal bundle. J Struct Biol 144, 195-208.