Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

Regulator of Awn Elongation 3, an E3 ubiquitin ligase, is responsible for loss of awns during African rice domestication.

Two species of rice have been independently domesticated from different ancestral wild species in Asia and Africa. Comparison of mutations that underlie phenotypic and physiological alterations associated with domestication traits in these species gives insights into the domestication history of rice in both regions. Asian cultivated rice, Oryza sativa, and African cultivated rice, Oryza glaberrima, have been modified and improved for common traits beneficial for humans, including erect plant architecture, nonshattering seeds, nonpigmented pericarp, and lack of awns. Independent mutations in orthologous genes associated with these traits have been documented in the two cultivated species. Contrary to this prevailing model, selection for awnlessness targeted different genes in O. sativa and O. glaberrima. We identify Regulator of Awn Elongation 3 (RAE3) a gene that encodes an E3 ubiquitin ligase and is responsible for the awnless phenotype only in O. glaberrima. A 48-bp deletion may disrupt the substrate recognition domain in RAE3 and diminish awn elongation. Sequencing analysis demonstrated low nucleotide diversity in a ~600-kb region around the derived rae3 allele on chromosome 6 in O. glaberrima compared with its wild progenitor. Identification of RAE3 sheds light on the molecular mechanism underlying awn development and provides an example of how selection on different genes can confer the same domestication phenotype in Asian and African rice.

Risk Factors Associated with Clinical Failure of Uterine Artery Embolization for Postpartum Hemorrhage.

PURPOSE: To identify risk factors for clinical failure of uterine artery embolization (UAE) for postpartum hemorrhage (PPH), with particular attention to the uterine artery diameter. MATERIALS AND METHODS: This retrospective study included 47 patients who underwent UAE for PPH between January 1, 2010, and January 31, 2021. Technical success was defined as the completion of embolization of the arteries thought to be the cause of the bleeding. Clinical success was defined as no recurrent bleeding or need for additional therapeutic interventions. Univariate and multivariate analyses were performed to examine the risk factors associated with clinical failure of UAE. RESULTS: Of the 47 patients, 6 had recurrent bleeding. Of the 6 patients, 4 underwent hysterectomy, and 2 underwent repeat embolization. The clinical success rate was 87.2% (41/47), with no major adverse events such as uterine infarction or death. In univariate analysis, there were slight differences in multiparity (P = .115) and placental abruption (P = .128) and a significant difference in the findings of a narrow uterine artery on digital subtraction angiography (DSA) (P = .005). In multivariate analysis, only a narrow uterine artery on DSA was a significant factor (odds ratio, 18.5; 95% confidence interval, 2.5-134.8; P = .004). CONCLUSIONS: A narrow uterine artery on DSA was a risk factor for clinically unsuccessful UAE for PPH. It may be prudent to conclude the procedure only after it is ensured that vasospasm has been relieved.

Molecular mechanism of internode elongation in rice.

Rice plants that form ventilated tissues, such as aerenchyma in the leaves, stems, and roots, allow for growth in waterlogged conditions (paddy fields), but they cannot breathe and drown in flooded environments where the whole plant body is submerged. However, deepwater rice plants grown in flood-prone areas of Southeast Asia survive in prolonged flooded environments by taking in air through an elongated stem (internode) and leaves that emerge above the water surface, even if the water level is several meters high and flooding continues for several months. Although it has been known that plant hormones, such as ethylene and gibberellins, promote internode elongation in deepwater rice plants, the genes that control rapid internode elongation during submergence have not been identified. We recently identified several genes responsible for the quantitative trait loci involved in internode elongation in deepwater rice. Identification of the the genes revealed a molecular gene network from ethylene to gibberellins in which internode elongation is promoted by novel ethylene-responsive factors and enhances gibberellin responsiveness at the internode. In addition, elucidation of the molecular mechanism of internode elongation in deepwater rice will help our understanding of the internode elongation mechanism in normal paddy rice and contribute to improving crops through the regulation of internode elongation.

Noninvasive imaging of hollow structures and gas movement revealed the gas partial-pressure-gradient-driven long-distance gas movement in the aerenchyma along the leaf blade to submerged organs in rice.

Rice (Oryza sativa) plants have porous or hollow organs consisting of aerenchyma, which is presumed to function as a low-resistance diffusion pathway for air to travel from the foliage above the water to submerged organs. However, gas movement in rice plants has yet to be visualized in real time. In this study involving partially submerged rice plants, the leaves emerging from the water were fed nitrogen-13-labeled nitrogen ([13 N]N2 ) tracer gas, and the gas movement downward along the leaf blade, leaf sheath, and internode over time was monitored. The [13 N]N2 gas arrived at the bottom of the plant within 10 min, which was 20 min earlier than carbon-11 photoassimilates. The [13 N]N2 gas movement was presumably mediated by diffusion along the aerenchyma network from the leaf blade to the root via nodes functioning as junctions, which were detected by X-ray computed tomography. These findings imply the diffusion of gas along the aerenchyma, which does not consume energy, has enabled plants to adapt to aquatic environments. Additionally, there were no major differences in [13 N]N2 gas movement between paddy rice and deepwater rice plants, indicative of a common aeration mechanism in the two varieties, despite the difference in their response to flooding.

Venous Sac and Feeding Artery Embolization versus Feeding Artery Embolization Alone for Treating Pulmonary Arteriovenous Malformations: Draining Vein Size Outcomes.

PURPOSE: To investigate and compare venous sac and feeding artery embolization (VFE) with feeding artery embolization (FAE) alone for treatment of pulmonary arteriovenous malformations (PAVMs), based on difference in outcomes in decrease of the size of the draining vein. MATERIALS AND METHODS: Twenty-six patients (7 male and 19 female; median age [interquartile range], 58 years [46-65 years]) with 42 simple PAVMs treated with coil embolization between August 2005 and December 2018 were retrospectively evaluated. Twenty PAVMs were treated with FAE early in the study period and compared with 22 PAVMs treated with VFE later in the study period. Follow-up computed tomography images obtained 8-20 months after embolotherapy were used for outcome analysis. Data related to patient demographics; follow-up period; baseline diameters of the feeding artery, venous sac, and draining vein; draining vein diameter after treatment; and decrease in the size of the draining vein, including the number reaching a threshold of 70% decrease, were compared between the 2 groups. RESULTS: The draining vein decreased in size by a median of 46.4% in the FAE group and 66.3% in the VFE group, and the difference between the 2 groups was statistically significant (P = .009). There were no significant differences in the other parameters. CONCLUSIONS: VFE leads to a greater decrease in the size of the draining vein than FAE, suggesting that VFE results in more complete occlusion than FAE for treatment of PAVMs.

Functional Domain Order of an Anti-EGFR × Anti-CD16 Bispecific Diabody Involving NK Cell Activation.

Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.

Diel O2 Dynamics in Partially and Completely Submerged Deepwater Rice: Leaf Gas Films Enhance Internodal O2 Status, Influence Gene Expression and Accelerate Stem Elongation for 'Snorkelling' during Submergence.

Deepwater rice has a remarkable shoot elongation response to partial submergence. Shoot elongation to maintain air-contact enables 'snorkelling' of O2 to submerged organs. Previous research has focused on partial submergence of deepwater rice. We tested the hypothesis that leaf gas films enhance internode O2 status and stem elongation of deepwater rice when completely submerged. Diel patterns of O2 partial pressure (pO2) were measured in internodes of deepwater rice when partially or completely submerged, and with or without gas films on leaves, for the completely submerged plants. We also took measurements for paddy rice. Deepwater rice elongated during complete submergence and the shoot tops emerged. Leaf gas films improved O2 entry during the night, preventing anoxia in stems, which is of importance for elongation of the submerged shoots. Expressions of O2 deprivation inducible genes were upregulated in completely submerged plants during the night, and more so when gas films were removed from the leaves. Diel O2 dynamics showed similar patterns in paddy and deepwater rice. We demonstrated that shoot tops in air enabled 'snorkelling' and increased O2 in internodes of both rice ecotypes; however, 'snorkelling' was achieved only by rapid shoot elongation by deepwater rice, but not by paddy rice.

Time-Course Transcriptomics Analysis Reveals Key Responses of Submerged Deepwater Rice to Flooding.

Water submergence is an environmental factor that limits plant growth and survival. Deepwater rice (Oryza sativa) adapts to submergence by rapidly elongating its internodes and thereby maintaining its leaves above the water surface. We performed a comparative RNA sequencing transcriptome analysis of the shoot base region, including basal nodes, internodes, and shoot apices of seedlings at two developmental stages from two varieties with contrasting deepwater growth responses. A transcriptomic comparison between deepwater rice cv C9285 and nondeepwater rice cv Taichung 65 revealed both similar and differential expression patterns between the two genotypes during submergence. The expression of genes related to gibberellin biosynthesis, trehalose biosynthesis, anaerobic fermentation, cell wall modification, and transcription factors that include ethylene-responsive factors was significantly different between the varieties. Interestingly, in both varieties, the jasmonic acid content at the shoot base decreased during submergence, while exogenous jasmonic acid inhibited submergence-induced internode elongation in cv C9285, suggesting that jasmonic acid plays a role in the submergence response of rice. Furthermore, a targeted de novo transcript assembly revealed transcripts that were specific to cv C9285, including submergence-induced biotic stress-related genes. Our multifaceted transcriptome approach using the rice shoot base region illustrates a differential response to submergence between deepwater and nondeepwater rice. Jasmonic acid metabolism appears to participate in the submergence-mediated internode elongation response of deepwater rice.

Loss of function at RAE2, a previously unidentified EPFL, is required for awnlessness in cultivated Asian rice.

Domestication of crops based on artificial selection has contributed numerous beneficial traits for agriculture. Wild characteristics such as red pericarp and seed shattering were lost in both Asian (Oryza sativa) and African (Oryza glaberrima) cultivated rice species as a result of human selection on common genes. Awnedness, in contrast, is a trait that has been lost in both cultivated species due to selection on different sets of genes. In a previous report, we revealed that at least three loci regulate awn development in rice; however, the molecular mechanism underlying awnlessness remains unknown. Here we isolate and characterize a previously unidentified EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family member named REGULATOR OF AWN ELONGATION 2 (RAE2) and identify one of its requisite processing enzymes, SUBTILISIN-LIKE PROTEASE 1 (SLP1). The RAE2 precursor is specifically cleaved by SLP1 in the rice spikelet, where the mature RAE2 peptide subsequently induces awn elongation. Analysis of RAE2 sequence diversity identified a highly variable GC-rich region harboring multiple independent mutations underlying protein-length variation that disrupt the function of the RAE2 protein and condition the awnless phenotype in Asian rice. Cultivated African rice, on the other hand, retained the functional RAE2 allele despite its awnless phenotype. Our findings illuminate the molecular function of RAE2 in awn development and shed light on the independent domestication histories of Asian and African cultivated rice.

Rice leaf hydrophobicity and gas films are conferred by a wax synthesis gene (LGF1) and contribute to flood tolerance.

Floods impede gas (O2 and CO2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas.

Computed tomography-guided preoperative localization of small lung nodules with indocyanine green.

Background Small, deep-seated lung nodules and sub-solid nodules are often difficult to locate without marking. Purpose To evaluate the success and complication rates associated with the use of indocyanine green (ICG) to localize pulmonary nodules before resection. Material and Methods This retrospective study was approved by our institutional review board. Informed consent for performing preoperative localization using ICG marking was obtained from all patients. Thirty-seven patients (14 men, 23 women; mean age = 63.1 years; age range = 10-82 years) with small peripheral pulmonary nodules underwent computed tomography (CT)-guided ICG marking immediately before surgery between March 2007 and June 2016. The procedural details and complication rates associated with ICG marking are described. Results The average nodule size and depth were 9.1 mm (range = 2-22 mm) and 9.9 mm (range = 0-33 mm), respectively. Marking was detected at the pleural surface in 35 patients (95%). Three cases of mild pneumothorax (8%), five cases of cough (14%), and one case of mild bloody sputum (3%) with no clinical significance were noted. There were no severe complications. The average duration required to perform the marking was 19.4 min (range = 12-41 min). Conclusion Our results indicate that CT-guided ICG marking is safe and useful for detecting the location of small pulmonary nodules preoperatively.

Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Hormone Distribution and Transcriptome Profiles in Bamboo Shoots Provide Insights on Bamboo Stem Emergence and Growth.

Growth and development are tightly co-ordinated events in the lifetime of living organisms. In temperate bamboo plants, spring is the season when environmental conditions are suitable for the emergence of new shoots. Previous studies demonstrated that bamboo plants undergo an energy-consuming 'fast stem growth' phase. However, the events during the initiation of stem elongation in bamboo are poorly understood. To understand the onset of bamboo stem growth, we performed hormone and transcriptome profiling of tissue regions in newly elongating shoots of the Moso bamboo Phyllostachys edulis. The growth hormones auxins, cytokinins and gibberellins accumulated in the shoot apex, while the stress hormones ABA, salicylic acid (SA) and jasmonic acid (JA) are predominantly found in the lower part of the stem. The mature basal part of the stem showed enrichment of transcripts associated with cell wall metabolism and biosynthesis of phenylpropanoid metabolites, such as lignin. In the young upper stem region, expression of cell formation- and DNA synthesis-related genes was enriched. Moreover, the apical region showed enhanced expression of genes involved in meristem maintenance, leaf differentiation and development, abaxial/adaxial polarity and flowering. Our findings integrate the spatial regulation of hormones and transcriptome programs during the initiation of bamboo stem growth.

Development of chromosome segment substitution lines harboring Oryza nivara genomic segments in Koshihikari and evaluation of yield-related traits.

Chromosome segment substitution lines (CSSLs) are rich genetic resources that can be mined for novel, agriculturally useful loci or that can be used directly as materials for breeding. To date, a number of rice CSSLs have been developed by crossing rice cultivars with its wild relatives as a means to tap into the potential of wild alleles in rice improvement. Oryza nivara is a wild relative of rice that is thought to be a progenitor of O. sativa spp. indica. In the present study, 26 CSSLs that covers the entire genome of O. nivara as contiguous, overlapping segments in the genomic background of a japonica cultivar, O. sativa cv. Koshihikari were developed. Evaluation of the CSSLs for several agriculturally important traits identified candidate chromosome segments that harbors QTLs associated with yield and yield-related traits. The results of the study revealed the potential of O. nivara as a source of novel alleles that can be used to improve the existing japonica cultivar.

The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water.

Living organisms must acquire new biological functions to adapt to changing and hostile environments. Deepwater rice has evolved and adapted to flooding by acquiring the ability to significantly elongate its internodes, which have hollow structures and function as snorkels to allow gas exchange with the atmosphere, and thus prevent drowning. Many physiological studies have shown that the phytohormones ethylene, gibberellin and abscisic acid are involved in this response, but the gene(s) responsible for this trait has not been identified. Here we show the molecular mechanism of deepwater response through the identification of the genes SNORKEL1 and SNORKEL2, which trigger deepwater response by encoding ethylene response factors involved in ethylene signalling. Under deepwater conditions, ethylene accumulates in the plant and induces expression of these two genes. The products of SNORKEL1 and SNORKEL2 then trigger remarkable internode elongation via gibberellin. We also demonstrate that the introduction of three quantitative trait loci from deepwater rice into non-deepwater rice enabled the latter to become deepwater rice. This discovery will contribute to rice breeding in lowland areas that are frequently flooded during the rainy season.

Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice.

Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice.

Enzymatic hydrolysis of nylons: quantification of the reaction rate of nylon hydrolase for thin-layered nylons.

Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M w = 22,900, M n = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis.

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αßßα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) from Arthrobacter sp. KI72.

Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylCp2) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylCp2 was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl and 25% glycerol. Diffraction data were collected from the native crystal to a resolution of 1.60 Å. The obtained crystal was spindle shaped and belonged to the C-centred orthorhombic space group C2221, with unit-cell parameters a=70.84, b=144.90, c=129.05 Å. A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.