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Structure of human complement C8, a precursor to membrane attack.

Bubeck D, Roversi P, Donev R, Morgan BP, Llorca O, Lea SM - J. Mol. Biol. (2010)

Bottom Line: C8 initiates membrane penetration and coordinates MAC pore formation.High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly.We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, UK.

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Two-dimensional images of negatively stained C8. C8 (2.5 μl of 0.03 mg/ml) was applied to a carbon-coated copper-palladium grid, glow-discharged for 10 s at 20 mA. Grids were negatively stained with 0.75% uranyl formate using the two-drop method.11 Images were taken under low-dose conditions (∼10 e−/Å2 per exposure) at a magnification of 59,000× on a Tecnai F30 microscope. Micrographs were digitized using a SCAI scanner (Z/I Imaging) at a step size of 7 μm and binned by a factor of 4, resulting in a pixel size of 4.74 Å/pixel (a). (b) 5167 windowed particles were subjected to 10 cycles of reference-free alignment using EMAN12 and classified into 362 classes. Representative 2D class averages indicate a wide range of orientations. The scale bar represents 110 Å.
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f0005: Two-dimensional images of negatively stained C8. C8 (2.5 μl of 0.03 mg/ml) was applied to a carbon-coated copper-palladium grid, glow-discharged for 10 s at 20 mA. Grids were negatively stained with 0.75% uranyl formate using the two-drop method.11 Images were taken under low-dose conditions (∼10 e−/Å2 per exposure) at a magnification of 59,000× on a Tecnai F30 microscope. Micrographs were digitized using a SCAI scanner (Z/I Imaging) at a step size of 7 μm and binned by a factor of 4, resulting in a pixel size of 4.74 Å/pixel (a). (b) 5167 windowed particles were subjected to 10 cycles of reference-free alignment using EMAN12 and classified into 362 classes. Representative 2D class averages indicate a wide range of orientations. The scale bar represents 110 Å.

Mentions: C8, isolated from plasma and purified under physiological conditions,10 is a stable, homogenous complex composed of three subunits, C8α, C8β, and C8γ, in which the intersubunit interactions remain intact (Fig. S1). Negatively stained C8 complexes were visualized by electron microscopy. Raw images (Fig. 1a) were aligned using reference-free alignment and classified into groups (Fig. 1b). Two-dimensional (2D) averages show that C8 has two distinct regions. The larger of the two appears globular and pseudo-2-fold symmetric with less density in the middle. The smaller one protrudes from this core.


Structure of human complement C8, a precursor to membrane attack.

Bubeck D, Roversi P, Donev R, Morgan BP, Llorca O, Lea SM - J. Mol. Biol. (2010)

Two-dimensional images of negatively stained C8. C8 (2.5 μl of 0.03 mg/ml) was applied to a carbon-coated copper-palladium grid, glow-discharged for 10 s at 20 mA. Grids were negatively stained with 0.75% uranyl formate using the two-drop method.11 Images were taken under low-dose conditions (∼10 e−/Å2 per exposure) at a magnification of 59,000× on a Tecnai F30 microscope. Micrographs were digitized using a SCAI scanner (Z/I Imaging) at a step size of 7 μm and binned by a factor of 4, resulting in a pixel size of 4.74 Å/pixel (a). (b) 5167 windowed particles were subjected to 10 cycles of reference-free alignment using EMAN12 and classified into 362 classes. Representative 2D class averages indicate a wide range of orientations. The scale bar represents 110 Å.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3021121&req=5

f0005: Two-dimensional images of negatively stained C8. C8 (2.5 μl of 0.03 mg/ml) was applied to a carbon-coated copper-palladium grid, glow-discharged for 10 s at 20 mA. Grids were negatively stained with 0.75% uranyl formate using the two-drop method.11 Images were taken under low-dose conditions (∼10 e−/Å2 per exposure) at a magnification of 59,000× on a Tecnai F30 microscope. Micrographs were digitized using a SCAI scanner (Z/I Imaging) at a step size of 7 μm and binned by a factor of 4, resulting in a pixel size of 4.74 Å/pixel (a). (b) 5167 windowed particles were subjected to 10 cycles of reference-free alignment using EMAN12 and classified into 362 classes. Representative 2D class averages indicate a wide range of orientations. The scale bar represents 110 Å.
Mentions: C8, isolated from plasma and purified under physiological conditions,10 is a stable, homogenous complex composed of three subunits, C8α, C8β, and C8γ, in which the intersubunit interactions remain intact (Fig. S1). Negatively stained C8 complexes were visualized by electron microscopy. Raw images (Fig. 1a) were aligned using reference-free alignment and classified into groups (Fig. 1b). Two-dimensional (2D) averages show that C8 has two distinct regions. The larger of the two appears globular and pseudo-2-fold symmetric with less density in the middle. The smaller one protrudes from this core.

Bottom Line: C8 initiates membrane penetration and coordinates MAC pore formation.High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly.We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density.

View Article: PubMed Central - PubMed

Affiliation: University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, UK.

Show MeSH
Related in: MedlinePlus