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Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143.

Kelleher A, Liu Z, Seid CA, Zhan B, Asojo OA - Acta Crystallogr F Struct Biol Commun (2015)

Bottom Line: The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%.LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement.All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

View Article: PubMed Central - HTML - PubMed

Affiliation: National School of Tropical Medicine, Baylor College of Medicine, 1102 Bates Avenue, Suite 550, Mail Stop BCM320, Houston, TX 77030-3411, USA.

ABSTRACT
Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59,000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Å resolution from orthorhombic crystals belonging to space group P2(1)2(1)2(1), with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%. LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement. All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

No MeSH data available.


Related in: MedlinePlus

(a) Typical crystals of LJL143 are thin, flat rods of less than 0.01 mm on the smallest face. (b) A sample diffraction image of LJL143 crystals reveals visible spots to 2.6 Å resolution as indicated by the arrow.
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fig2: (a) Typical crystals of LJL143 are thin, flat rods of less than 0.01 mm on the smallest face. (b) A sample diffraction image of LJL143 crystals reveals visible spots to 2.6 Å resolution as indicated by the arrow.

Mentions: The typical yield of LJL143 was 50 mg per litre of fermentation supernatant. The resulting protein had an electrophoretic mobility of 42 kDa. The predicted theoretical molecular mass of LJL143 is 33 594.92 Da. LJL143 has two predicted N-glycosylation sites at 42NQTH and 241NKTC, which may contribute to the almost 10 kDa increase in molecular mass. LJL143 crystals were grown using different polyethylene glycols (PEG) as precipitants. The best diffracting crystals were obtained after 48 h in the conditions detailed in Table 2 ▸. These crystals are illustrated in Fig. 2 ▸(a). Diffraction images reveal visible diffraction spots beyond 2.6 Å resolution (Fig. 2 ▸b). The crystal belonged to the orthorhombic space group P212121, with approximate unit-cell parameters a = 57.5, b = 70.2, c = 79.5 Å (Table 3 ▸). Based on the estimated Matthews coefficient and solvent-content prediction (http://www.ruppweb.org/Mattprob), a monomer is expected in the asymmetric unit (Matthews, 1968 ▸; Kantardjieff & Rupp, 2003 ▸). This corresponds to a unit-cell volume of 321 104 Å3 (with an asymmetric unit of 80 276 Å3), a Matthews coefficient of 2.39 Å3 Da−1 and an estimated solvent content of 48.5%.


Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143.

Kelleher A, Liu Z, Seid CA, Zhan B, Asojo OA - Acta Crystallogr F Struct Biol Commun (2015)

(a) Typical crystals of LJL143 are thin, flat rods of less than 0.01 mm on the smallest face. (b) A sample diffraction image of LJL143 crystals reveals visible spots to 2.6 Å resolution as indicated by the arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498716&req=5

fig2: (a) Typical crystals of LJL143 are thin, flat rods of less than 0.01 mm on the smallest face. (b) A sample diffraction image of LJL143 crystals reveals visible spots to 2.6 Å resolution as indicated by the arrow.
Mentions: The typical yield of LJL143 was 50 mg per litre of fermentation supernatant. The resulting protein had an electrophoretic mobility of 42 kDa. The predicted theoretical molecular mass of LJL143 is 33 594.92 Da. LJL143 has two predicted N-glycosylation sites at 42NQTH and 241NKTC, which may contribute to the almost 10 kDa increase in molecular mass. LJL143 crystals were grown using different polyethylene glycols (PEG) as precipitants. The best diffracting crystals were obtained after 48 h in the conditions detailed in Table 2 ▸. These crystals are illustrated in Fig. 2 ▸(a). Diffraction images reveal visible diffraction spots beyond 2.6 Å resolution (Fig. 2 ▸b). The crystal belonged to the orthorhombic space group P212121, with approximate unit-cell parameters a = 57.5, b = 70.2, c = 79.5 Å (Table 3 ▸). Based on the estimated Matthews coefficient and solvent-content prediction (http://www.ruppweb.org/Mattprob), a monomer is expected in the asymmetric unit (Matthews, 1968 ▸; Kantardjieff & Rupp, 2003 ▸). This corresponds to a unit-cell volume of 321 104 Å3 (with an asymmetric unit of 80 276 Å3), a Matthews coefficient of 2.39 Å3 Da−1 and an estimated solvent content of 48.5%.

Bottom Line: The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%.LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement.All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

View Article: PubMed Central - HTML - PubMed

Affiliation: National School of Tropical Medicine, Baylor College of Medicine, 1102 Bates Avenue, Suite 550, Mail Stop BCM320, Houston, TX 77030-3411, USA.

ABSTRACT
Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59,000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Å resolution from orthorhombic crystals belonging to space group P2(1)2(1)2(1), with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%. LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement. All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

No MeSH data available.


Related in: MedlinePlus