Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

Sociocultural dilemma of Japanese steeplejacks

Japanese steeplejacks are good at working in high places as construction workers, and they have been called tobi for a longtime. They now play an important role in completing modern civil engineering projects and in the construction of high-rise buildings; however, their lifestyle is considered by most to be quaint but outdated. Originally, they were unskilled workmen at construction sites. In the 18th century, they were engaged in repairing houses or setting up scaffolding, helping carpenters, but they worked as firefighters whenever fires broke out. Their traditional work system did not change throughout the Meiji era, although Japanese society became greatly modernized. After World War II, the industrialization of Japanese society required highly developed technology in civil engineering and architecture. This provided an opportunity for them to establish their positions as trained professional workers. However, the number of skilled tobi professionals has continued to decrease because the younger generation does not consider this profession desirable career. Improving not only the professional skills but also the way of living to the extent as a modern high-tech society demands will be the key for the tobi's work system to become attractive.     

Linkage map of phenotype and RFLP markers in rice

The results from linkage mapping activities at Kyushu University during the last 10 years are summarized in this paper. The present paper concisely reveals present situation on linkage map of phenotype markers, the integration linkage map of phenotype and RFLP markers and the genetic stocks available. Some of the problems in this field, in addition, are pointed out and discussed.     

Mutations of the Btk gene in 12 unrelated families with X-linked agammaglobulinemia in Japan

Alterations of the Bruton's tyrosine kinase(Btk) gene are responsible for X-linked agammaglobulinemia (XLA). Although mutations in various regions were reported mainly in the Caucasian population, correlation between the locations of mutation and the clinical phenotypes remains unclear. We report 12 abnormalities of theBtk gene found in 12 unrelated families out of 14 XLA families in Japan and their clinical features. We utilized Southern blotting and single-strand conformation polymorphism (SSCP) analysis. Gene rearrangement in the kinase domain was identified in two patients by Southern blotting. Seven point mutations, two small deletions, and one small insertion were detected by SSCP and sequencing. The SSCP analysis also provided information about the carriers in these families. We found some clinical heterogeneity in the affected family members with the same gene mutation. Moreover, there is considerable inconsistency between the locations of gene aberrations and the immunological phenotypes. Some patients with a nonsense mutation, which may result in the lack of kinase domain, have detectable B cells and immunoglobulins. These identified alterations will provide valuable clues to theBtk protein function and the pathogenesis of XLA.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens?

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.     

Evidence for the involvement of jasmonic acid in the control of the stem-growth habit of soybean plants

Soybean plants show diversity in stem-growth habit which ranges from the determinate type to the indeterminate type. Stem growth of determinate plants abruptly terminate near the beginning of flowering. The possible involvement of jasmonic acid (JA) in the control of the stem growth-habit was examined in indeterminate and determinate isolines of soybean [ Glycine max (L.) Merril cv. Harosoy]. JA-like activities in leaves of both isolines were very low 20 days before the commencement of flowering. The activity increased rapidly thereafter and reached a maximum near the time of flowering. Although the activities in leaves of both isolines fluctuated in a similar manner, the activity in the determinate isoline was much higher than that in the indeterminate isoline after flowering. The presence of JA in the leaves of the determinate isoline was confirmed by purification by high-performance liquid chromatography and by mass spectrometry. Exogenous application of JA to cultured shoot apices of the indeterminate isoline strongly inhibited growth. These results suggest that jasmonic acid is a major endogenous factor that controls the growth habit of soybean plants.     

The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)     

A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1.

Furin, a subtilisin-like mammalian endoprotease, is thought to be responsible for the processing of many proprotein precursors of cellular and viral origin, including gp160 of human immunodeficiency virus type 1, which share the consensus processing site motif, Arg-X-Lys/Arg-Arg, for protease recognition (for reviews, see P. J. Barr, Cell 66:1-3, 1991, and Y. Nagai, Trends Microbiol. 1:81-87, 1993). To confirm and extend the concept that gp160 is processed by furin, we used here a cell line, LoVo, which was recently demonstrated to be furin defective. Unexpectedly, LoVo cells were found to process gp160 as efficiently as normal cell lines do, hence being able to fuse with CD4-expressing HeLa cells and to produce fully infectious virions. On the other hand, the same cell line was almost totally incapable of processing Newcastle disease virus fusion glycoprotein with a similar oligobasic cleavage recognition motif, providing a strong case for furin-mediated processing. Our present study thus raises a further need to search for and identify the proteinases involved in human immunodeficiency virus type 1 gp160 processing rather than supporting the notion that furin is responsible.