Computational protocol: Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function

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Protocol publication

[…] C. albicans FKBP12 was crystallized in three different space groups, P1, P21212, and C2, by hanging-drop vapor diffusion. The P1 crystal form was obtained by using a mixture of 25% polyethylene glycol (PEG) 3000, 0.1 M NaCl, and 0.1 M Tris-HCl (pH 8.0) as a crystallization reagent. The P21212 form grew in a solution of 2 M ammonium sulfate and 0.1 M citrate (pH 5.5), and the C2 crystal form was crystallized in a mixture of 25% PEG 8000, 0.1 M sodium acetate (pH 4.5), and 0.2 M lithium sulfate. Each hanging drop contained a 1:1 ratio of ~10 mg/ml protein to crystallization reagent. The crystallization reagent supplemented with 25% glycerol was used as a cryosolvent. For cryopreservation, the crystals were dipped for several seconds in the cryosolvent prior to their placement in the liquid nitrogen stream. The C. albicans FKBP12(P104G)-FK506 complex crystallized in the P21 space group in a mixture of 2 M ammonium sulfate, 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)-NaOH (pH 10.5), and 0.2 M lithium sulfate. One millimole of FK506 was added to the protein prior to crystallization. A solution of 4 M ammonium sulfate was used as a cryoprotectant. A. fumigatus FKBP12 at a concentration of 20 mg/ml was crystallized in the P212121 space group in a mixture of 2 M ammonium sulfate, 0.1 M Tris-HCl (pH 7.0), and 0.2 M lithium sulfate. A 4 M ammonium sulfate solution was used as a cryoprotectant. A. fumigatus FKBP12(P90G)-FK506 crystals were obtained by mixing 20 mg/ml protein (with 1 mM FK506) with a solution containing 2 M lithium sulfate, 0.1 M Tris-HCl (pH 8.5), and 2% PEG 400 at a 1:1 ratio. C. albicans FKBP12(P104G) apo crystals were grown by mixing protein at 10 mg/ml 1:1 with a combination of 2 M ammonium sulfate, 0.1 M CAPS (pH 10.5), and 0.2 M lithium sulfate. The crystals could be cryopreserved straight from the drop. Data were collected at Advanced Light Source beamline 8.3.1 or Advanced Photon Source. Data were processed with HKL3000 or MOSFLM. Initial phases were obtained in each case by using molecular replacement (MolRep), starting with the human FKBP12 model (PDB code 2PPN). The A. fumigatus and C. glabrata apo structures were solved by using the C. albicans apo high-resolution structure as a starting model in MolRep. The FK506-bound structures were solved by using the requisite apo structures as starting models. All model building was carried out with COOT or O, and refinements were performed in PHENIX (). […]

Pipeline specifications

Software tools iMosflm, Molrep, Coot, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Candida albicans, [Candida] glabrata, Aspergillus fumigatus
Diseases Mycoses
Chemicals Cysteine, Tacrolimus