A combinatorial approach to crystallization of PX-BAR unit of the human Sorting Nexin 9.
Sorting nexins (SNXs) form a family of proteins known to interact with endosomal vesicles and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the interface of endocytic, actin polymerizing, and signal transduction events in the cell. Here we report crystallization of the SNX9 PX-BAR domain protein. Initially we used an ordinary protein construct design, and protein crystallization approaches resulted in obtaining granular crystal-like precipitation. SDS-PAGE and MS analysis of the crystal-like precipitation followed by protein construct optimization and using of macro seeding technique resulted in X-ray quality diffracting crystals. The crystals belonged to P2(1)2(1)2(1) space group (a=65.6 A, b=117.5 A, c=145.8 A) with two protein molecules per asymmetric unit. A complete SAD data set from Se-Methionine derived crystal (3.2 A) has been collected to solve the structure.
Max-Planck-Institute for Molecular Physiology, Department of Physical Biochemistry, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Olena
O
Pylypenko
Alexander
A
Ignatev
Richard
R
Lundmark
Erika
E
Rasmuson
Sven R
SR
Carlsson
Alexey
A
Rak
D002352:Carrier Proteins / Q000737:chemistry*; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins / Q000737:chemistry*; D014961:X-Ray Diffraction
D002352:Carrier Proteins; C400533:SNX9 protein, human; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins