Dataset Information

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.

SUBMITTER: Harada A

PROVIDER: S-EPMC5137467 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

Harada Ayaka A Sato Yukari Y Kamimura Naofumi N Venugopalan Nagarajan N Masai Eiji E Senda Toshiya T

Acta crystallographica. Section F, Structural biology communications 20161130 Pt 12

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 ...[more]

PMID: 27917838

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Project description:BACKGROUND: Polyploidy is important from a phylogenetic perspective because of its immense past impact on evolution and its potential future impact on diversification, survival and adaptation, especially in plants. Molecular population genetics studies of polyploid organisms have been difficult because of problems in sequencing multiple-copy nuclear genes using Sanger sequencing. This paper describes a method for sequencing a barcoded mixture of targeted gene regions using next-generation sequencing methods to overcome these problems. RESULTS: Using 64 3-bp barcodes, we successfully sequenced three chloroplast and two nuclear gene regions (each of which contained two gene copies with up to two alleles per individual) in a total of 60 individuals across 11 species of Australian Poa grasses. This method had high replicability, a low sequencing error rate (after appropriate quality control) and a low rate of missing data. Eighty-eight percent of the 320 gene/individual combinations produced sequence reads, and >80% of individuals produced sufficient reads to detect all four possible nuclear alleles of the homeologous nuclear loci with 95% probability.We applied this method to a group of sympatric Australian alpine Poa species, which we discovered to share an allopolyploid ancestor with a group of American Poa species. All markers revealed extensive allele sharing among the Australian species and so we recommend that the current taxonomy be re-examined. We also detected hypermutation in the trnH-psbA marker, suggesting it should not be used as a land plant barcode region. Some markers indicated differentiation between Tasmanian and mainland samples. Significant positive spatial genetic structure was detected at <100 km with chloroplast but not nuclear markers, which may be a result of restricted seed flow and long-distance pollen flow in this wind-pollinated group. CONCLUSIONS: Our results demonstrate that 454 sequencing of barcoded amplicon mixtures can be used to reliably sample all alleles of homeologous loci in polyploid species and successfully investigate phylogenetic relationships among species, as well as to investigate phylogeographic hypotheses. This next-generation sequencing method is more affordable than and at least as reliable as bacterial cloning. It could be applied to any experiment involving sequencing of amplicon mixtures.

Dataset Information

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

Publications

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

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