Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family.
The NAC domain (residues 1-168) of ANAC, encoded by the abscisic acid-responsive NAC gene from Arabidopsis thaliana, was recombinantly produced in Escherichia coli and crystallized in hanging drops. Three morphologically different crystal forms were obtained within a relatively narrow range of conditions: 10-15% PEG 4000 and 0.1 M imidazole/malic acid buffer pH 7.0 in the reservoir, 3.2-7.7 mg ml(-1) protein stock and a 1:1 ratio of reservoir to protein solution in the hanging drop. One of the crystal forms, designated crystal form III, was found to be suitable for further X-ray analysis. Form III crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.0, b = 75.2, c = 80.8 A at 100 K. The unit-cell volume is consistent with two molecules in the asymmetric unit and a peak in the native Patterson map suggests the presence of a non-crystallographic twofold axis parallel to a crystallographic axis. Size-exclusion chromatography of the NAC domain showed that the dimeric state is also the preferred state in solution and probably represents the biologically active form. Data sets were collected from four potential heavy-atom derivatives of the form III crystals. The derivatized crystals are reasonably isomorphous with the non-derivatized crystals and the four data sets are being evaluated for use in structure determination by multiple isomorphous replacement.
Olsen
Addie Nina
AN
Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark.
Ernst
Heidi Asschenfeldt
HA
Lo Leggio
Leila
L
Johansson
Eva
E
Larsen
Sine
S
Skriver
Karen
K
D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
C474015:ANAC protein, Arabidopsis; D029681:Arabidopsis Proteins; D018744:DNA, Plant; D011994:Recombinant Proteins; D014157:Transcription Factors