Probing the structure of F-actin: cross-links constrain atomic models and modify actin dynamics 1 1Edited by M. F. Moody

Orlova, Albina; Galkin, Vitold E.; VanLoock, Margaret S.; Kim, Eldar; Shvetsov, Alexander; Reisler, Emil; Egelman, Edward H.

doi:10.1006/jmbi.2001.4945

Cited by 56 publications

(49 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The F-actin model produced here is also consistent with density maps generated for F-actin from a variety of experimental methods. The agreement with the map of Orlova et al (14) is shown in Fig. 4.…”

Section: Discussionsupporting

confidence: 88%

“…The inhibited in vitro sliding observed in these filaments is most likely due to impediments to the structural transitions that are needed for force generation (20). Indeed, EM reconstruction of similarly cross-linked actin filaments (by a disulfide bond between Cys-41 and Cys-374 in a yeast actin mutant) revealed only minor structural perturbations (14). ANP cross-linking was therefore an attractive approach to the crystallization of nativelike longitudinal actin dimers.…”

Section: Results and Analysismentioning

confidence: 99%

“…The first structural model of F-actin was obtained by Holmes et al (11), by using fiber-diffraction data extending to 8.4-Å resolution to determine the approximate orientations and positions of actin protomers in the filament. The F-actin filament has been analyzed in numerous subsequent imaging studies, by electron microscopy (EM) with negative staining and cryoelectron microscopy (12)(13)(14)(15)(16)(17). These imaging studies have supported the basic features of the Holmes model and have led to structural refinements and variations under differing circumstances and conditions.…”

mentioning

confidence: 99%

See 2 more Smart Citations

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

Kudryashov

Sawaya

Adisetiyo

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The 2.5-Å resolution crystal structure is reported for an actin dimer, composed of two protomers cross-linked along the longitudinal (or vertical) direction of the F-actin filament. The crystal structure provides an atomic resolution view of a molecular interface between actin protomers, which we argue represents a near-native interaction in the F-actin filament. The interaction involves subdomains 3 and 4 from distinct protomers. The atomic positions in the interface visualized differ by 5-10 Å from those suggested by previous models of F-actin. Such differences fall within the range of uncertainties allowed by the fiber diffraction and electron microscopy methods on which previous models have been based. In the crystal, the translational arrangement of protomers lacks the slow twist found in native filaments. A plausible model of F-actin can be constructed by reintroducing the known filament twist, without disturbing significantly the interface observed in the actin dimer crystal.actin crystal structure ͉ F-actin filament ͉ motor proteins A ctin is one of the most highly conserved proteins in nature, where it plays key roles in contraction, cell shape and motility, and subcellular organization. Actin's myriad functions are regulated by protein-protein interactions. Besides interacting with an enormously diverse set of other cellular proteins (1), actin's most critical functions arise from its interactions with itself as it assembles to form F-actin filaments. Because actin carries out its cellular functions through its filamentous form, knowing the detailed structure of actin filaments is an important step in achieving a mechanistic understanding of actin function.Our understanding of actin has been advanced greatly by intensive investigation into its three-dimensional (3D) structure. Because the filamentous form of actin is essentially incompatible with the growth of 3D crystals, crystallographic studies have focused on actin in its monomeric form. The 3D structure of monomeric actin (G-actin) was determined first by Kabsch et al.(2) as a complex, with DNase I serving as a polymerization inhibitor. The crystal structure of monomeric actin has since been determined at atomic resolution under a variety of conditions, varying, for example, in actin isoform (3), the identity of the bound nucleotide (4, 5), and in the identity of the other proteins (6, 7) or small molecules added as polymerization inhibitors (8, 9). As a consequence, the structure of the actin monomer (G-actin) is understood in considerable detail, although some important issues remain open regarding nucleotide-dependent changes in G-actin structure, including a possible transition between the open and closed nucleotide cleft conformations and the order-disorder shift of the DNase I-binding loop (4,7,10).Structural models of the actin filament have derived mainly from methods other than crystallography. The first structural model of F-actin was obtained by Holmes et al. (11), by using fiber-diffraction data extending to 8.4-Å resolution to determi...

show abstract

Section: Discussionsupporting

confidence: 88%

Section: Results and Analysismentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

Kudryashov

Sawaya

Adisetiyo

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…As the difference between these two is a 57°O rotation, there is no way that a similar interface can exist between subunits in ParM and Factin. Whereas F-actin has substantial longitudinal contacts between subdomain 2 (corresponding to subdomain Ib in ParM) and the subunit above it along the same long-pitch helix [12][13][14] , in ParM no such contacts exist. The most substantial interface in the ParM filament involves subdomain IIb (corresponding to subdomain 4 in F-actin) with subdomain Ia of the subunit above it (Fig.…”

Section: Parm and F-actin Have Different Subunit-subunit Interfacesmentioning

confidence: 99%

The structure of bacterial ParM filaments

Orlova

Garner

Galkin

et al. 2007

Nat Struct Mol Biol

Self Cite

View full text Add to dashboard Cite

Bacterial ParM is a homolog of eukaryotic actin and is involved in moving plasmids so that they segregate properly during cell division. Using cryo-EM and three-dimensional reconstruction, we show that ParM filaments have a different structure from F-actin, with very different subunitsubunit interfaces. These interfaces result in the helical handedness of the ParM filament being opposite to that of F-actin. Like F-actin, ParM filaments have a variable twist, and we show that this involves domain-domain rotations within the ParM subunit. The present results yield new insights into polymorphisms within F-actin, as well as the evolution of polymer families.ParM is a bacterial actin homolog involved in plasmid segregation 1,2 . The structures of both ParM and MreB 3 , another bacterial actin homolog, as well as FtsZ 4 , a tubulin homolog, show that the eukaryotic cytoskeleton is similar to a prokaryotic cytoskeleton that has only recently been studied. Although the higher-order structure of FtsZ is still unknown, it has been assumed that both ParM and MreB assemble into filaments with an arrangement of subunits very similar to that found in F-actin. However, a crystal structure of monomeric ParM has unexpectedly shown that its main differences from actin are in regions expected to be involved in the filamentous subunit-subunit interface 2 . A previous study 2 has used EM of negatively stained ParM filaments to generate a low-resolution three-dimensional reconstruction, which suggests that the ParM filament is very similar to F-actin.We have used negative staining, cryo-EM and quick-freeze/deep-etch EM to examine filaments formed by the ParM protein from Escherichia coli. The question that we intended to answer was how similar to F-actin the ParM filaments appear at higher resolution. We show that the ParM filaments are substantially different from F-actin, with a very different subunit-subunit interface. Because of the very different interface, the helical handedness of the ParM filaments is opposite to that of F-actin. RESULTS Variable twist in ParM filamentsEM shows that ParM forms long filaments when incubated with the nonhydrolyzable ATP analog AMP-PNP (Fig. 1). We analyzed both negatively stained (Fig. 1a) and frozenhydrated ParM filaments (Fig. 1b), but took advantage of the iterative helical real-space reconstruction (IHRSR) method 5 to surmount the problems posed by flexible and disordered filaments. In this method, rather than individual filaments being treated as ideal helices with a uniform structure, short segments are analyzed, classified and reconstructed separately. One of the most obvious forms of disorder revealed by such a method is the presence of variable twist in the ParM filaments, even greater than what has been shown for F-actin 6-8 . The variability in average twist angles found in unstained frozen-hydrated ParM segments (28,886 segments, each segment ~480 Å long) is shown in Figure 2a. This variability arises from different segments within the same filaments, rather than different ...

show abstract

“…On the other hand, it has been known that due to the cylindrical averaging and overlap of different Bessel functions in x-ray fiber diffraction, models derived from this technique may not be unique, either. For example, a fiber diffraction model for F-actin [31] provided "an almost perfect fit" to the observed x-ray diffraction pattern, but has subsequently been shown to have little experimental support [32]. Hopefully, higher resolution EM reconstructions of such filamentous phages as fd will be able to resolve this controversy generated by the different models from NMR, x-ray fiber diffraction and EM.…”

Section: Structural Heterogeneitymentioning

confidence: 99%

Single-particle reconstruction from EM images of helical filaments

Egelman

2007

Current Opinion in Structural Biology

Self Cite

View full text Add to dashboard Cite

SummaryHelical filaments were the first structures to be reconstructed in three-dimensions from electron microscopic images, and continue to be extensively studied due to the large number of such helical polymers found in biology. In principle, a single image of a helical polymer provides all of the different projections needed to reconstruct the three-dimensional structure. Unfortunately, many helical filaments have been refractory to the application of traditional (Fourier-Bessel) methods due to variability, heterogeneity and weak scattering. Over the past several years many of these problems have been surmounted using single-particle type approaches that can do substantially better than Fourier-Bessel approaches. Applications of these new methods to viruses, actin filaments, pili and many other polymers show the great advantages of the new methods.

show abstract

Probing the structure of F-actin: cross-links constrain atomic models and modify actin dynamics 1 1Edited by M. F. Moody

Cited by 56 publications

References 37 publications

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

The structure of bacterial ParM filaments

Single-particle reconstruction from EM images of helical filaments

Contact Info

Product

Resources

About