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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: Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector.C8 initiates membrane penetration and coordinates MAC pore formation.Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.

View Article: PubMed Central - PubMed

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

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Related in: MedlinePlus

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: Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector.C8 initiates membrane penetration and coordinates MAC pore formation.Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus