title: Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.
abstract: A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P3121 or P3221 crystals, which diffracted to 2.5-3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P3121/P3221 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Å resolution and belonged to space group P21212, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P21212 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.
authors.affiliation: Department of Accelerator Science, School of High Energy Accelerator Science, SOKENDAI (The Graduate University for Advanced Studies), 1-1 Oho, Tsukuba, Ibaraki 305-0031, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Harada
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Naofumi
authors.initials: N
authors.lastname: Kamimura
authors.affiliation: GM/CA@APS, Argonne National Laboratory, Lemont, IL 60439, USA.
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Venugopalan
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Masai
authors.affiliation: Department of Accelerator Science, School of High Energy Accelerator Science, SOKENDAI (The Graduate University for Advanced Studies), 1-1 Oho, Tsukuba, Ibaraki 305-0031, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Senda
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042301:Sphingomonadaceae / Q000737:chemistry* / Q000201:enzymology; D013763:Tetrahydrofolates / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D010090:Oxidoreductases, O-Demethylating
keywords: O-demethylase; Sphingobium sp. SYK-6; cryoprotectant; hemihedral twinning; microseeding; tetrahydrofolate