U-shape rotating anti-cathode compact X-ray generator: 20 times stronger than the commercially available X-ray source.

A new type of U-shape anti-cathode X-ray generator in which the inner surface of a cylindrical target is irradiated by an electron beam has been made by modifying a conventional rotating anti-cathode X-ray generator whose brightness in the catalog is 12 kW mm(-2). The target material (Cu), target radius (50 mm) and rotating speed (6,000 r.p.m.) were not changed in this modification. A brightness of 52 kW mm(-2) was obtained by this U-shape-type X-ray generator. This means that the brightness of the new type is 4.3 times greater than that of the old unmodified one. Furthermore, the new-type X-ray generator yielded a brightness of 129 kW mm(-2) by adding a carbon coating on the Cu target. This means an overall increase of brightness of ten times. The original generator has the highest brightness in the generators of the same class (having a radius of 50 mm and rotation speed of 6,000 r.p.m.). Observations showed that Cu Kα counts at vertical incidence of the electron beam onto the surface of the new target, which is initially optically smooth, decrease as the surface is roughened by a severe thermal stress caused by strong electron beam exposure. Further observation reveals, however, that oblique incidence of the electron beam onto the roughened surface drastically increased the X-ray output and amounts to twice as much as that from a smooth surface at vertical incidence. Thus, at the present stage, an overall increase of brightness has been realised at a level 20 times stronger than that of the original commercially offered X-ray generator that we modified.

Highly bright X-ray generator using heat of fusion with a specially designed rotating anticathode.

A new type of rotating anticathode X-ray generator has been developed, in which the electron beam irradiates the inner surface of a U-shaped anticathode (Cu). A high-flux electron beam is focused on the inner surface by optimizing the shape of the bending magnet. The power of the electron beam can be increased to the point at which the irradiated part of the inner surface is melted, because a strong centrifugal force fixes the melted part on the inner surface. When the irradiated part is melted, a large amount of energy is stored as the heat of fusion, resulting in emission of X-rays 4.3 times more brilliant than can be attained by a conventional rotating anticathode. Oscillating translation of the irradiated position on the inner surface during use is expected to be very advantageous for extending the target life. A carbon film coating on the inner surface is considered to suppress evaporation of the target metal and will be an important technique in further realization of highly bright X-ray generation.

Conceptual design of novel IP-conveyor-belt Weissenberg-mode data-collection system with multi-readers for macromolecular crystallography. A comparison between Galaxy and Super Galaxy.

Galaxy is a Weissenberg-type high-speed high-resolution and highly accurate fully automatic data-collection system using two cylindrical IP-cassettes each with a radius of 400 mm and a width of 450 mm. It was originally developed for static three-dimensional analysis using X-ray diffraction and was installed on bending-magnet beamline BL6C at the Photon Factory. It was found, however, that Galaxy was also very useful for time-resolved protein crystallography on a time scale of minutes. This has prompted us to design a new IP-conveyor-belt Weissenberg-mode data-collection system called Super Galaxy for time-resolved crystallography with improved time and crystallographic resolution over that achievable with Galaxy. Super Galaxy was designed with a half-cylinder-shaped cassette with a radius of 420 mm and a width of 690 mm. Using 1.0 A incident X-rays, these dimensions correspond to a maximum resolutions of 0.71 A in the vertical direction and 1.58 A in the horizontal. Upper and lower screens can be used to set the frame size of the recorded image. This function is useful not only to reduce the frame exchange time but also to save disk space on the data server. The use of an IP-conveyor-belt and many IP-readers make Super Galaxy well suited for time-resolved, monochromatic X-ray crystallography at a very intense third-generation SR beamline. Here, Galaxy and a conceptual design for Super Galaxy are described, and their suitability for use as data-collection systems for macromolecular time-resolved monochromatic X-ray crystallography are compared.

Large-format imaging plate and weissenberg camera for accurate protein crystallographic data collection using synchrotron radiation.

Off-line and on-line protein data-collection systems using an imaging plate as a detector are described and their components reported. The off-line scanner IPR4080 was developed for a large-format imaging plate ;BASIII' of dimensions 400 x 400 mm and 400 x 800 mm. The characteristics of this scanner are a dynamic range of 10(5) photons pixel(-1), low background noise and high sensitivity. A means of reducing electronic noise and a method for finding the origin of the noise are discussed in detail. A dedicated screenless Weissenberg camera matching IPR4080 with synchrotron radiation was developed and installed on beamline BL6B at the Photon Factory. This camera can attach one or two sheets of 400 x 800 mm large-format imaging plate inside the film cassette by evacuation. The positional reproducibility of the imaging plate on the cassette is so good that the data can be processed by batch job. Data of 93% completeness up to 1.6 A resolution were collected on a single axis rotation and the value of R(merge) becomes 4% from a tetragonal lysozyme crystal using a set of two imaging-plate sheets. Comparing two types of imaging plates, the signal-to-noise ratio of the ST-VIP-type imaging plate is 25% better than that of the BASIII-type imaging plate for protein data collection using 1.0 and 0.7 A X-rays. A new on-line protein data-collection system with imaging plates is specially designed to use synchrotron radiation X-rays at maximum efficiency.

Time-resolved protein crystallography with large-angle oscillations: an application of a protein data-collection system using the Weissenberg technique and a large-format imaging plate.

A diffraction-intensity data-collection system with synchrotron radiation X-rays utilizing the screenless Weissenberg technique and incorporating a large-format imaging plate is one of the most suitable apparatus for time-resolved protein crystallography with larger angle oscillations than hitherto described. The time resolution and data quality of the system have been tested using a tetragonal lysozyme crystal as a test sample in a flow-cell experiment at the bending-magnet beamline 18B at the Photon Factory, and a time resolution of 15 min is confirmed.

A preliminary description of the crystal structure of gamma-glutamyltranspeptidase from E. coli K-12.

The structure of GGT [EC 2.3.2.2] from E. coli K-12 was studied at 3 A resolution by X-ray crystallography. Initial protein phases were calculated using two kinds of Pb2+ derivatives. The phases were refined by non-crystallographic 2-fold symmetry electron density averaging combined with solvent flattening and histogram matching. The GGT molecule has overall dimensions of 60 x 50 x 40 A. There are two antiparallel beta-pleated sheets consisting of 6 and 7 beta-strands. The two beta-sheets form a wall-like structure. Twelve short alpha-helices were detected, of which the maximum length appears to be four helix turns.

Crystallization and preliminary X-ray analysis of gamma-glutamyltranspeptidase from Escherichia coli K-12.

gamma-Glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12 has been purified and crystallized by means of vapor diffusion in hanging drops. Two kinds of crystals on cell dimensions were found for X-ray diffraction analysis, one from ammonium sulfate and the other from polyethylene glycol 6000 as precipitants. The crystals of the orthorhombic form grown in the presence of 15% polyethylene glycol and 20 mM sodium acetate buffer were chosen for further analysis. The crystals belonged to space group P2(1)2(1)2(1), with cell dimensions of a = 128.1, b = 129.9 and c = 79.2 A, and two molecules constitute an asymmetric unit. These crystals diffracted to 2.0 A resolution and were suitable for X-ray crystallographic studies.

Electrocardiogram studies in llamas.

Electrocardiograms (ECG) of 3 captive llamas (Lama glama) were recorded by a telemeter at a farm in Japan. The pattern of the ECGs was similar to the ruminant pattern in the AB lead position. QRS and T-waves were discordant in polarity except in one llama, where the polarity of the T wave changed according to the HR. In the quiet state, the HR varied between 60-80/min depending on the nervousness of the llama. After running and after being held, the HR increased to more than 100/min. During the recordings there were some variations of the HR which could be due to respiratory arrhythmia. Second-degree AV block and supraventricular premature complexes were also found in two llamas.

Crystal structure analysis of omega-amino acid:pyruvate aminotransferase with a newly developed Weissenberg camera and an imaging plate using synchrotron radiation.

The three-dimensional structure of omega-amino acid:pyruvate aminotransferase from Pseudomonas sp. F-126, an isologous alpha 4 tetramer containing pyridoxal 5'-phosphate (PLP) as a cofactor, has been determined at 2.0 A resolution. The diffraction data were collected with a newly developed Weissenberg camera with a Fuji Imaging Plate, using synchrotron radiation. The mean figure-of-merit was 0.57. The subunit is rich in secondary structure and comprises two domains. PLP is located in the large domain. The high homology in the secondary structure between this enzyme and aspartate aminotransferase strongly indicates that these two types of enzymes have evolved from a common ancestor.

X-ray diffraction photographs of chicken gizzard G-actin.DNase I complex crystals taken with synchrotron radiation.

X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 degrees oscillation range were recorded on a single film up to 3.5 A resolution.

Crystallographic studies of the chicken gizzard G-actin X DNase I complex at 5A resolution.

The structure of the chicken gizzard G-actin X DNase I complex has been determined at 5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the "cypress wood" models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.

Power spectral analysis of auditory brain stem responses to pure tone stimuli.

Power spectral analysis and digital filtration of the auditory brain stem responses to tone burst stimuli were carried out in three adult subjects with normal hearing. Three dominant peaks (peaks A. B, C), ranging in frequency 50-150, 500-600, and 1000-1100 Hz, were observed in the spectrograms of the responses to 4 kHz at 80 dB SL. Power of higher frequency components (peaks B and C) decreased markedly or disappeared completely with decreased intensity as well as decreased frequency of stimulus, while peak A around 100 Hz consistently appeared regardless of stimulus intensity or frequency. Main spectral components of the individual waves in the ABR were determined as follows: peak A for the slow positive deflection in the response, peak B for waves VI and VII, peak C for waves II and IV, and peaks B and C for waves I, III and V.

Power spectral analysis of auditory evoked response.

Five normal hearing adults were examined for power spectral analysis of AER to 1000-Hz pure tone. Major frequency components of responses to stimuli at 50 dB hearing level exist at 1 to 14 c/s. The power spectra of responses to stimuli at 10 dB hearing level are greater than those of the SBA in the frequency components between 3 and 9 c/s; furthermore, the total power of 3 to 9 c/s indicates a clear difference between the 2. Incidentally, at 512-msec analysis time, the total power of 3 to 9 c/s of the AER are significantly greater than that at 1024-msec analysis time, whereas those of the SBA show little discrepancy at the 2; thus at 512-msec analysis time, the total power of 4 to 9 c/s yields a clear difference between the AER and the SBA responses averaged over 50 stimuli even at 10 dB hearing level.

Crystallization and preliminary crystallographic data of chicken gizzard G-actin . DNase I complex and Physarum G-actin . DNase I complex.

Smooth muscle G-actin from chicken gizzard and Physarum plasmodium G-actin both interact with DNase I and form 1 : 1 complexes. These complexes were crystallized by using polyethylene glycol 6000 as a precipitant. Both crystals belong to the same orthorhombic space group P2(1)2(1)2(1). The cell dimensions of chicken gizzard G-actin.DNase I complex are a=42.00 +/- 0.07 A, b=225.3 +/- 0.4 A, and c=77.4 +/- 0.1 A, while those of Physarum G-actin.DNase I complex are a=42 A, b=221 A, and c=77 A.