Expression, purification and crystallization of the Atg5-Atg16 complex essential for autophagy.
Atg5 is a novel 34 kDa protein that is covalently modified by Atg12, a ubiquitin-like modifier, and forms a complex with Atg16. The Atg12-Atg5-Atg16 complex localizes to autophagosome precursors and plays an essential role in autophagosome formation. Saccharomyces cerevisiae Atg5 in complex with the N-terminal regions of Atg16 was expressed, purified and crystallized in four crystal forms. Forms I, II and III belong to space group P2(1), with unit-cell parameters a = 66.3, b = 104.4, c = 112.1 A, beta = 92.1 degrees (form I), a = 79.5, b = 101.4, c = 95.1 A, beta = 98.6 degrees (form II) or a = 56.9, b = 101.2, c = 66.5 A, beta = 100.6 degrees (form III). Form IV belongs to space group P4(2)2(1)2, with unit-cell parameters a = 73.3, c = 148.1 A. Diffraction data were collected from all crystal forms and high-resolution data to beyond 2.0 A resolution were obtained from a form IV crystal.
Matsushita
Minako
M
Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-12, W-6, Kita-ku, Sapporo 060-0812, Japan.
Suzuki
Nobuo N
NN
Fujioka
Yuko
Y
Ohsumi
Yoshinori
Y
Inagaki
Fuyuhiko
F
D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
C498649:ATG16 protein, S cerevisiae; C102268:ATG5 protein, S cerevisiae; D000071187:Autophagy-Related Protein 5; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases