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Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

Jagadeesan G, Malathy P, Gunasekaran K, Harikrishna Etti S, Aravindhan S - Acta Crystallogr F Struct Biol Commun (2014)

Bottom Line: Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates.In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins.Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Presidency College, Chennai 600 005, India.

ABSTRACT
Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

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Three-dimensional single crystals of cormorant haemoglobin.
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fig2: Three-dimensional single crystals of cormorant haemoglobin.

Mentions: Crystals were obtained by the hanging-drop vapour-diffusion method at 18°C. Polyethylene glycol (PEG) with different molecular weights was initially used to screen the crystallization conditions. It was subsequently found that a combination of PEG 3350 and sodium chloride was suitable for obtaining multiple microcrystal clusters. Single crystals were separated from the microcrystal clusters and immediately flash-cooled in liquid nitrogen, but diffracted poorly with streaky spots at very low resolution. Good crystals suitable for X-ray diffraction were grown after 25 d at 18°C using 25% PEG 3350, 10% glycerol, 0.5 M NaCl, 50 mM sodium phosphate buffer pH 7.5 equilibrated against 3 µl protein solution and 3 µl reservoir solution (Fig. 2 ▶). The Hb crystals were mounted in a cryoloop and data were collected at cryotemperature using a MAR345 imaging plate at the Central Leather Research Institute (CLRI), Chennai, India. A total of 108 frames were collected at 18°C using a crystal-to-detector distance of 100 mm, an oscillation angle of 1° and an exposure time of 300 s per image; the crystal diffracted to a maximum resolution of 3.5 Å (Fig. 3 ▶). Intensity measurements were processed and analyzed using iMosflm (Battye et al., 2011 ▶). The data-collection and refinement statistics are summarized in Table 1 ▶.


Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

Jagadeesan G, Malathy P, Gunasekaran K, Harikrishna Etti S, Aravindhan S - Acta Crystallogr F Struct Biol Commun (2014)

Three-dimensional single crystals of cormorant haemoglobin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231857&req=5

fig2: Three-dimensional single crystals of cormorant haemoglobin.
Mentions: Crystals were obtained by the hanging-drop vapour-diffusion method at 18°C. Polyethylene glycol (PEG) with different molecular weights was initially used to screen the crystallization conditions. It was subsequently found that a combination of PEG 3350 and sodium chloride was suitable for obtaining multiple microcrystal clusters. Single crystals were separated from the microcrystal clusters and immediately flash-cooled in liquid nitrogen, but diffracted poorly with streaky spots at very low resolution. Good crystals suitable for X-ray diffraction were grown after 25 d at 18°C using 25% PEG 3350, 10% glycerol, 0.5 M NaCl, 50 mM sodium phosphate buffer pH 7.5 equilibrated against 3 µl protein solution and 3 µl reservoir solution (Fig. 2 ▶). The Hb crystals were mounted in a cryoloop and data were collected at cryotemperature using a MAR345 imaging plate at the Central Leather Research Institute (CLRI), Chennai, India. A total of 108 frames were collected at 18°C using a crystal-to-detector distance of 100 mm, an oscillation angle of 1° and an exposure time of 300 s per image; the crystal diffracted to a maximum resolution of 3.5 Å (Fig. 3 ▶). Intensity measurements were processed and analyzed using iMosflm (Battye et al., 2011 ▶). The data-collection and refinement statistics are summarized in Table 1 ▶.

Bottom Line: Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates.In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins.Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Presidency College, Chennai 600 005, India.

ABSTRACT
Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

Show MeSH
Related in: MedlinePlus