Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy

Edstrom, Ronald D.; Meinke, Marilyn H.; Yang, Xu; Yang, Ruiguo; Evans, D. F.

doi:10.1021/bi00438a004

Cited by 39 publications

(18 citation statements)

References 16 publications

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“…Not only has the number of bridges in the PhK complex been previously questioned, but even their very existence, especially since these structural components were not observed by either STM or AFM (Edstrom et al 1989, 1990). Furthermore, with negatively stained PhK, the question has been raised of whether the formation of pseudo‐bridges or the distortion of genuine bridges might occur as artifacts of specimen preparation (Norcum et al 1994).…”

Section: Resultsmentioning

confidence: 99%

Cryoelectron microscopy reveals new features in the three‐dimensional structure of phosphorylase kinase

Nadeau¹

2005

Protein Science

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Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (␣␤␥␦) 4 . PhK comprises two arched octameric (␣␤␥␦) 2 lobes that are oriented back-to-back with overall D 2 symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Threedimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 Å. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90°instead of 68°, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.Keywords: phosphorylase kinase; cryoelectron microscopy; three-dimensional reconstruction; single particle analysis; structural analysis Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is in skeletal muscle a hexadecameric complex composed of four copies of four different subunits: (␣␤␥␦) 4 (for review, see Brushia and Walsh 1999). Within the PhK complex, the activity of the catalytic ␥ subunit is tightly controlled by the regulatory ␣, ␤, and ␦ (endogenous calmodulin) subunits; occupancy of or phosphorylation of allosteric sites on those regulatory subunits leads to PhK's activation. Because of its large mass, 1.3 MDa, the PhK complex is particularly amenable to analysis by microscopic techniques, and has been studied by transmission (TEM) (Cohen 1974;Schramm and Jennissen 1985;Trempe et al. 1986;Norcum et al. 1994;Wilkinson et al. 1994Wilkinson et al. , 1997, scanning transmission (STEM) (Trempe et al. 1986;Norcum et al. 1994;Wilkinson et al. 1994), and scanning tunneling electron microscopy (STM) (Edstrom et al. 1989(Edstrom et al. , 1990 Abbreviations: AFM, atomic force microscopy; EM, electron microscopy; ns, negative stain; PhK, phosphorylase kinase; SAXS, small-angle X-ray scattering; STEM, scanning transmission EM; STM, scanning tunneli...

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Section: Resultsmentioning

confidence: 99%

Cryoelectron microscopy reveals new features in the three‐dimensional structure of phosphorylase kinase

Nadeau¹

2005

Protein Science

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“…The first such method, scanning tunneling microscopy (STM), allows high resolution, but it is limited to conducting substrates, and imaging in the presence of an aqueous and hence conductive solvent presents a challenge. Thus, most early studies were of dried protein layers, and their focus was on the qualitative appearance of individual molecules rather than on the structure of the adsorbed layers (3,4). Atomic force microscopy (AFM) overcomes the two principal problems of STM mentioned above, but it is more intrusive and can lead to "sweeping" of molecules from the surface (5), unless the molecules are covalently immobilized (6).…”

Section: Introductionmentioning

confidence: 99%

Adsorbed Layers of Ferritin at Solid and Fluid Interfaces Studied by Atomic Force Microscopy

Johnson

Yuan

Lenhoff

2000

Journal of Colloid and Interface Science

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“…The slightly lower values of the dried samples (85 ± 11% of the control) may be due to small changes in structure of the protein or to incomplete solubilization of the AChR from the HOPG surface by the detergent. DISCUSSION STM imaging of biological macromolecules has only recently been applied to nucleic acids (22)(23)(24)(25), globular proteins (26,27), and glycogen (28) …”

Section: %32mentioning

confidence: 99%

Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor.

Bertazzon¹,

Conti‐Tronconi²,

Raftery³

1992

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

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Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy

Cited by 39 publications

References 16 publications

Cryoelectron microscopy reveals new features in the three‐dimensional structure of phosphorylase kinase

Cryoelectron microscopy reveals new features in the three‐dimensional structure of phosphorylase kinase

Adsorbed Layers of Ferritin at Solid and Fluid Interfaces Studied by Atomic Force Microscopy

Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor.

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