Integrated metabolomics and transcriptomics analysis of roots of Bupleurum chinense and B. scorzonerifolium, two sources of medicinal Chaihu.

Radix Bupleuri (Chaihu in Chinese) is a traditional Chinese medicine commonly used to treat colds and fevers. The root metabolome and transcriptome of two cultivars of B. chinense (BCYC and BCZC) and one of B. scorzonerifolium (BSHC) were determined and analyzed. Compared with BSHC, 135 and 194 differential metabolites were identified in BCYC and BCZC, respectively, which were mainly fatty acyls, organooxygen metabolites. A total of 163 differential metabolites were obtained between BCYC and BCZC, including phenolic acids and lipids. Compared with BSHC, 6557 and 5621 differential expression genes (DEGs) were found in BCYC and BSHC, respectively, which were annotated into biosynthesis of unsaturated fatty acid and fatty acid metabolism. A total of 4,880 DEGs existed between the two cultivars of B. chinense. The abundance of flavonoids in B. scorzonerifolium was higher than that of B. chinense, with the latter having higher saikosaponin A and saikosaponin D than the former. Pinobanksin was the most major flavonoid which differ between the two cultivars of B. chinense. The expression of chalcone synthase gene was dramatically differential, which had a positive correlation with the biosynthesis of pinobanksin. The present study laid a foundation for further research on biosynthesis of flavonoids and terpenoids of Bupleurum L.

A chromosome-level genome assembly for Dracaena cochinchinensis reveals the molecular basis of its longevity and formation of dragon's blood.

Dracaena, a remarkably long-lived and slowly maturing species of plant, is world famous for its ability to produce dragon's blood, a precious traditional medicine used by different cultures since ancient times. However, there is no detailed and high-quality genome available for this species at present; thus, the molecular mechanisms that underlie its important traits are largely unknown. These factors seriously limit the protection and regeneration of this rare and endangered plant resource. Here, we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level. The D. cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31 619 predicted protein-coding genes. Analysis showed that D. cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions. The expansion of two gene classes, cis-zeatin O-glucosyltransferase and small auxin upregulated RNA, were found to account for its longevity and slow growth. Two transcription factors (bHLH and MYB) were found to be core regulators of the flavonoid biosynthesis pathway, and reactive oxygen species were identified as the specific signaling molecules responsible for the injury-induced formation of dragon's blood. Our study provides high-quality genomic information relating to D. cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon's blood. These findings will facilitate resource protection and sustainable utilization of Dracaena.

Identification of the original plants of cultivated Bupleuri Radix based on DNA barcoding and chloroplast genome analysis.

Bupleuri Radix is the dry root of certain species of the genus Bupleurum and is commonly used in traditional Chinese medicine. The increasing global demand for Bupleuri Radix cannot be fulfilled with wild populations only. Therefore, cultivated Bupleurum is now the main commercial source of this medicinal product. Different species of Bupleurum show different medicinal properties and clinical effects, making reliable authentication and assignment of correct botanical origin for medicinal species critical. However, accurate identification of the cultivated Bupleurum species is difficult due to dramatic morphological variations resulting from cultivation. In this study, we sampled 56 cultivated Bupleurum populations of six different morphotypes (Types A-F) from the main production areas of China, and 10 wild populations of four species were used as reference materials. Conventional DNA barcoding was conducted to identify cultivated Bupleurum species. Additionally, verification based on complete chloroplast genomes was performed and new chloroplast markers were developed and evaluated. The combination of these methods resulted in the successful identification of all cultivated Bupleurum individuals. Three chloroplast regions are recommended as additional barcodes for the genus: ycf4_cemA, psaJ_rpl33, and ndhE_ndhG. This is a reliable and promising strategy that can be applied to the authentication of natural products and the identification of other medicinal plant species with similar taxonomic problems.

Overexpression of BcERF3 increases the biosynthesis of saikosaponins in Bupleurum chinense.

Chaihu, the dried roots of some species of Bupleurum L., is a famous Chinese herbal medicine for treatment of liver- and cold-related diseases, in which saikosaponins (SSs) are the major active compounds. Many of the genetic components upstream of SS biosynthetic pathways have been characterized; however, the regulatory mechanisms remain elusive. In this study we identified the APETALA2/Ethylene Responsive Factor family transcription factor gene BcERF3 from B. chinense. The expression of BcERF3 was induced in methyl-jasmonate-treated adventitious root of B. chinense; it was also expressed at higher levels in roots than in other tissues (stem, leaf, flower, and tender fruit of early fruiting plants). Transient expression of BcERF3 in the leaves of Nicotiana benthamiana resulted in intracellular localization of the protein in the nucleus. It was also demonstrated that the number of SSs was greater in BcERF3-overexpressing hairy roots of B. chinense than in plants treated with empty vector controls. This coincided with upregulation of ß-AS, which encodes a key enzyme involved with triterpenoid biosynthesis. In conclusion, BcERF3 plays a positive regulatory role in the biosynthesis of SSs.

New Advances of CRISPR/Cas9 Technique and its Application in Disease Treatment and Medicinal Plants Research.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology is widely used in disease treatment and medicinal plant improvements due to its advantages, such as easy operation, low time consumption, and high efficiency. However, potential off-target risks still exist in practical applications and need to be solved urgently. OBJECTIVES: This study aimed to review the application progress of CRISPR/Cas9 technology in the field of disease treatment and medicinal agriculture in recent years. Furthermore, the study discusses the ways to reduce the off-target effect of CRISPR/Cas9 technology, providing a reference for the further application of this technology. METHODS: CiteSpace software was used to analyze relevant literature data from 2013 to August 2021, and search results were retrieved from Web of Science, PubMed, and CNKI databases. RESULTS: In the field of disease treatment, CRISPR/Cas9 technology has great potential to cure challenging human diseases and has been widely used in drug target development, drug design, and screening. In crop breeding, CRISPR/Cas9 accelerates the improvement of crop varieties and shortens the number of years of crop breeding. By adjusting the length and GC content of sgRNA and changing the concentration of Cas9/sgRNA complex to reduce the off-target effect of CRISPR/Cas9 technology, the target genes can be manipulated more accurately. CONCLUSION: CRISPR/Cas9 technology is an indispensable and key technology in the field of disease treatment and medicinal plants. With the in-depth study of the off-target effect, CRISPR/Cas9 technology can have broader application prospects in the fields of medicine and medicinal agriculture.

Analysis of unigenes involved in lateral root development in Bupleurum chinense and B. scorzonerifolium.

MAIN CONCLUSION: We identified IAA13 negatively associated with lateral root number by comparing the differential expressed genes between Bupleurum chinense and B. scorzonerifolium. Dried roots of the genus Bupleurum L. are used as a herbal medicine for diseases in Asia. Bupleurum chinense has a greater number of lateral roots than B. scorzonerifolium, but the genetic mechanisms for such differences are largely unknown. We (a) compared the transcriptome profiles of the two species and (b) identified a subset of candidate genes involved in auxin signal transduction and explored their functions in lateral root development. By isoform sequencing (Iso-Seq) analyses of the whole plant, more unigenes were found in B. scorzonerifolium (118,868) than in B. chinense (93,485). Given the overarching role of indole-3-acetic acid (IAA) as one of the major regulators of lateral root development, we identified 539 unigenes associated with auxin signal transduction. Fourteen and 44 unigenes in the pathway were differentially expressed in B. chinense and B. scorzonerifolium, respectively, and 3 unigenes (LAX2, LAX4, and IAA13) were expressed in both species. The number of lateral root primordia increased after exogenous auxin application at 8 h and 12 h in B. scorzonerifolium and B. chinense, respectively. Since overexpression of IAA13 in Arabidopsis reduced the number of lateral roots, we hypothesized that IAA13 is involved in the reduction of the number of lateral roots in B. scorzonerifolium.

Recent Progress in Saikosaponin Biosynthesis in Bupleurum.

BACKGROUND: Chaihu is a popular traditional Chinese medicine that has been used for centuries. It is traditionally used to treat cold fever and liver-related diseases. Saikosaponins (SSs) are one of the main active components of chaihu, in addition to essential oils, flavonoids, and polysaccharides. Considerable effort is needed to reveal the biosynthesis and regulation of SSs on the basis of current progress. OBJECTIVE: The aim of this study is to provide a reference for further studies and arouse attention by summarizing the recent achievements of SS biosynthesis. METHODS: All the data compiled and presented here were obtained from various online resources, such as PubMed Scopus and Baidu Scholar in Chinese, up to October 2019. RESULTS: A few genes of the enzymes of SSs participating in the biosynthesis of SSs were isolated. Among these genes, only the P450 gene was verified to catalyze the SS skeleton ß-amyrin synthase. Several UDP-glycosyltransferase genes were predicted to be involved in the biosynthesis of SSs. SSs could be largely biosynthesized in the phloem and then transported from the protoplasm, which is the biosynthetic site, to the vacuoles to avoid self-poisoning. As for the other secondary metabolites, the biosynthesis of SSs was strongly affected by environmental factors and the different species belonging to the genus of Bupleurum. Transcriptional regulation was studied at the molecular level. CONCLUSION: Profound discoveries in SSs may elucidate the mechanism of diverse the monomer formation of SSs and provide a reference for maintaining the stability of SS content in Radix Bupleuri.

Differential Expression of Genes Involved in Saikosaponin Biosynthesis Between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd.

Radix Bupleuri (roots of Bupleurum spp.) is an important medicinal herb. Triterpenoid saponins of saikosaponins generally constitute the main class of secondary metabolites of plants in the Bupleurum genus. However, the molecular regulatory mechanism underlying their biosynthesis remains elusive. In this study, we observed significantly different saikosaponin biosynthesis between Bupleurum chinense and Bupleurum scorzonerifolium at the seedling stage. The sequential and expression characterization of 232 genes in the triterpenoid saponin biosynthetic pathway, which includes the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway, between B. chinense and B. scorzonerifolium was also investigated. Sixty of these genes may be involved in saikosaponin biosynthesis. Manipulation of these genes, especially those of the ß-AS, P450, and UGT families, may improve saikosaponin production.

Prevalence and prognostic impact of comorbidities and peripheral blood indices in sarcomas.

BACKGROUND: The prognostic impact of comorbidities in patients with sarcomas is not well defined. The aims of this study were to examine the implications of comorbidities and abnormal peripheral blood indices in patients with sarcomas. METHODS: A population-based database was assembled to extract patients with sarcoma in Hong Kong between January 2004 and March 2018. Charlson's Comorbidity Index (CCI) score and prevalence of comorbidities, neutrophil, lymphocyte and platelet counts at diagnosis were assessed. The prognostic values of CCI, neutrophil-lymphocyte (NLR) and platelet-lymphocyte ratios (PLR) were estimated using Cox proportional hazard models. Restricted cubic spline plots were used to explore the association of baseline NLR and PLR with all-cause and cancer-specific mortality. RESULTS: Among 3358 eligible patients with sarcomas, 52.2% died after a median 26 months of follow-up. The most common comorbidities were diabetes mellitus (9.8%) and cerebrovascular disease (4.8%). Patients with higher CCI had higher mortality (CCI=3 vs CCI=2; HR 1.49; 95% CI 1.19 to 1.87; p<0.01; CCI ≥7 vs CCI =2; HR 3.20; 95% CI 2.62 to 3.92; p<0.001). Abnormal NLR and PLR levels were associated with higher all-cause mortality (NLR: HR 1.698, p<0.001, 95% CI 1.424 to 2.025; PLR: HR 1.346, p<0.001, 95% CI 1.164 to 1.555) and cancer-related mortality (NLR: HR 1.648, p<0.001, 95% CI 1.341 to 2.024; PLR: HR 1.430, p<0.001, 95% CI 1.205 to 1.697). CONCLUSIONS: This is the largest population-based soft-tissue or bone sarcoma cohort worldwide. Comorbidities have significant negative prognostic impact on the survival of patients with sarcomas. Moreover, NLR and PLR are robust prognostic factors, and abnormal NLR and PLR have negative effects yet non-linear effects on survival.

[Genome survey analysis and SSR loci mining of Bupleurum falcatum].

Buplewrum falcatum is a traditional Chinese medicine,which is mainly used for the treatment of cold and liver protection. B. falcatum is dominantly cultivated in Japan as well as planted in China,Korea and other countries and regions. In order to determine the appropriate sequencing strategy,the genome survey before large-scale genome sequencing is needed. This survey can provide information about the size and complexity of the whole genome of the target species. In the present study,the next generation sequencing technology( Illumina Hiseq 2000) was used to analyze the genome size and complexity of B. falcatum. In addition,SSR loci were analyzed from the sequenced data. Primer 3 was used to design specific primers and 33 pairs of primers were randomly selected for PCR with template DNA of B. falcatum,and the PCR system and optimal annealing temperature were screened. A total of 288. 64 G genome sequence data was obtained,and the estimated genome size of B. falcatum was 2 119. 58 Mb. The measured genome data depth was138×; the rate of heterozygosity was 1. 84%; and the ratio of repeat sequence was 83. 89%. It is speculated that the genome of B. falcatum is complex. The preliminary assembly was performed with K-mer = 41,and the contig N50 was 224 bp,the total length 896. 97 Mb,the scaffold N50 313 bp,and the total length was 922. 67 Mb. A total of 91 377 SSR sequences were detected in the sequenced genome data which were distributed in 70 809 unigenes.The main type is dinucleotide repeats,with 49 680 sequences,accounting for70. 16%. Among the 33 pairs of primers randomly synthesized according to the obtained SSR sequences,21 pairs were successfully amplifying the target sequences. The results will be helpful for later large scale genome sequencing and SSR molecular markers development for germplasm identification and trait mapping.

SlMYC1 Regulates Type VI Glandular Trichome Formation and Terpene Biosynthesis in Tomato Glandular Cells.

Tomato (Solanum lycopersicum) glandular trichomes function as biochemical factories that synthesize a diverse array of specialized metabolites. Terpenoids are the most diverse class of plant specialized metabolites, with volatile mono- and sesquiterpenes playing important roles in plant defense. Although the biosynthetic pathways of volatile terpenes in tomato glandular trichomes have been well described, little is known about their regulation. Here, we demonstrate that SlMYC1, a basic helix-loop-helix transcription factor, differentially regulates mono- and sesquiterpene biosynthesis in the type VI glandular trichomes of tomato leaves and stems. SlMYC1 functions as a positive regulator of monoterpene biosynthesis in both leaf and stem trichomes but as a negative regulator of sesquiterpene biosynthesis in stem trichomes. SlMYC1 is also essential for type VI glandular trichome development, as knocking down SlMYC1 led to the production of smaller type VI glandular trichomes at lower densities, and knocking out this gene led to their absence. Our findings reveal a role for SlMYC1 not only in type VI glandular trichome development but also in the regulation of terpene biosynthesis in tomato.

Therapeutic Strategies in EGFR Mutant Non-Small Cell Lung Cancer.

OPINION STATEMENT: Non-small cell lung cancer (NSCLC) harboring epidermal growth receptor (EGFR) mutation has distinct genomic characteristics. Introduction of systemic treatments that specifically targeted actionable EGFR mutations has changed the therapeutic paradigm in this group of patients. Moreover, newer generations of EGFR tyrosine-kinase inhibitors (EGFR-TKIs) with superior pharmacokinetics and pharmacodynamics properties such as dacomitinib and osimertinib, when used in the front-line setting, have shown more favorable treatment outcomes than first-generation EGFR-TKIs. In addition, evolving molecular technologies such as droplet digital polymerase chain reaction (ddPCR) and next-generation sequencing (NGS) has enhanced our understanding towards the genetics and epigenetics in pathogenesis, drug-resistant mechanisms as well as improved diagnostic accuracy and efficacy. On the other hand, the recent development in immunotherapies has pushed anti-cancer treatment to new frontiers in many cancers including lung cancer. While ongoing research are focusing on how benefits of immunotherapy can be potentiated, the combinational use of EGFR-TKIs and checkpoint inhibitors have been shown repeatedly in prior trials to cause significant toxicities. This approach cannot be recommended outside of a clinical trial at this time. Overall, remarkable progresses have opened new therapeutic strategies with which patient survival is further improved. In this review, we shall discuss the latest treatment strategies in EGFR mutation positive NSCLC with a focus on latest evidence, and how advances in molecular diagnostics can play an important role patient management.

Trunk surface agarwood-inducing technique with Rigidoporus vinctus: An efficient novel method for agarwood production.

Only when Aquilaria spp. or Gyrinops spp. trees are wounded, due to insect attack, or microbial invasion, agarwood can be successfully induced. In the present study, a fungus which can induce agarwood formation efficiently was isolated and a suitable method for its application to induce agarwood formation was developed. Rigidoporus vinctus was isolated from the inner layers from infectious A. sinensis trees. When the fermentation liquid of fungi inoculated back to A. sinensis tree, agarwood was found to be induced. In addition, a novel method called trunk surface agarwood-inducing technique (Agar-Sit) was developed to produce agarwood with R. vinctus. The alcohol soluble extract content of the agarwood, up to 38.9%, far higher than the requirement (10%) in Chinese Pharmacopoeia and the six characteristic compounds of agarwood used as Chinese Medicinal Materials were all detected. Their relative percentages of the sesquiterpenes in the essential oil were 22.76%. This is the first report of the Agar-Sit and also the application of R. vinctus in agarwood induction. According to the results, when the combination of Agar-Sit and R. vinctus is used agarwood can be induced with high yield and good quality.

Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg.

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

Application of CRISPR/Cas9 in plant biology.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.

Agarwood Formation Induced by Fermentation Liquid of Lasiodiplodia theobromae, the Dominating Fungus in Wounded Wood of Aquilaria sinensis.

Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fungi isolated from an agarwood-producing A. sinensis tree and subsequently identify the fungi capable of promoting agarwood formation. We identified a total of 110 fungi isolates based on their morphological characteristics and rDNA ITS sequences. These isolates came from four different layers (namely the decomposing layer, agarwood layer, transition layer, and normal layer) near the agarwood formation site of the trunk. According to the experimental results, most of them belonged to Dothideomycetes (81.82%), while the others to Sordariomycetes (13.64%) or Eurotiomycetes (4.55%). Of note, 88 isolates were shown belonging to the species of Lasiodiplodia theobromae that are most frequently isolated from different layers. In addition, when the fermentation liquid of two isolates of L. theobromae (AF4 and AF12) and one isolate of Fusarium solani (AF21) was inoculated into the A. sinensis wood using the Agar-Wit technique, promoted agarwood formation was observed; however, the effect of AF21 did not keep stable in the later test, while AF4 and AF12 still functioned 1 year later. This study may lay a foundation for exploring the underlying mechanism of agarwood formation as well as fungi application in agarwood production.

Zhongdanyaozhi No. 1 and Zhongdanyaozhi No. 2 Are Hybrid Cultivars of Salvia miltiorrhiza with High Yield and Active Compounds Content.

Salvia miltiorrhiza Bunge is an important medicinal plant used for the treatment of cardiovascular disease. Intraspecific hybridization between a male sterile line and inbred lines was followed by 39 F1 crossings. Cultivars "Zhongdanyaozhi No. 1" (ZD1) and "Zhongdanyaozhi No. 2" (ZD2) were obtained. In 2012 and 2013 tests in Beijing, the two cultivars were compared with three widely accepted types, SDCK, SXCK and HNCK from Shandong, Shanxi and Henan provinces. The yield of ZD1 and ZD2 exceeded the three CKs by more than 48.2% and 39.2%, respectively; the composition of the two hybrid cultivars was similar to the control, although the content of some compounds varied to some extent. The content of salvianolic acid B and tanshinone II A of both ZD1 and ZD2 could measure up the requirement of Chinese Pharmacopoeia. The former showed no obvious advantage than the three CKs, while the later's tanshinone II A was 29.6% higher than the three CKs. Taken together, ZD1 is a high yielding and thick-root-type cultivar which is suitable for decoction pieces; while ZD2 is suitable for component especially lipophilic component extraction. ZD1 and ZD2 reported here are the first cultivars obtained by the hybridization of S. miltiorrhiza.

Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis.

Agarwood, a highly valuable resinous and fragrant heartwood of Aquilaria plants, is widely used in traditional medicines, incense and perfume. Only when Aquilaria trees are wounded by external stimuli do they form agarwood sesquiterpene defensive compounds. Therefore, understanding the signaling pathway of wound-induced agarwood formation is important. Jasmonic acid (JA) is a well-characterized molecule that mediates a plant's defense response and secondary metabolism. However, little is known about the function of endogenous JA in agarwood sesquiterpene biosynthesis. Here, we report that heat shock can up-regulate the expression of genes in JA signaling pathway, induce JA production and the accumulation of agarwood sesquiterpene in A. sinensis cell suspension cultures. A specific inhibitor of JA, nordihydroguaiaretic acid (NDGA), could block the JA signaling pathway and reduce the accumulation of sesquiterpene compounds. Additionally, compared to SA and H2O2, exogenously supplied methyl jasmonate has the strongest stimulation effect on the production of sesquiterpene compounds. These results clearly demonstrate the central induction role of JA in heat-shock-induced sesquiterpene production in A. sinensis.

Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2.

Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Šresolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = ß = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.