Michael Molitsky

The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results

The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5-0.6 A wavelength) with fluxes up to 8-18 x 10(12) photons s(-1) (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-maximum) at the sample position can be varied from 2.2 mm x 1.0 mm (horizontal x vertical, unfocused) to 0.083 mm x 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a kappa-geometry goniometer and beamline control software allows optimal strategies to be adopted in protein crystallographic experiments, thus maximizing the chances of their success. A large-area mosaic 3 x 3 CCD detector allows high-quality diffraction data to be measured rapidly to the crystal diffraction limits. The beamline layout and the X-ray optical and endstation components are described in detail, and the results of representative crystallographic experiments are presented.

A new detector for time-resolved small angle X-ray scattering studies

A new detector for time-resolved small-angle X-ray scattering has been designed and built for experiments at the Advanced Photon Source of Argonne National Laboratory. This detector is made from a 500 mum thick by 150 mm diameter ultra-high purity silicon wafer, which directly converts X-rays into electron-hole pairs. The electrodes are concentric rings that integrate the scattered X-rays over the azimuthal angle. The widths of the rings are optimized for the size of the X-ray beam and its energy spread. Only 128 rings, or channels, are needed to measure a scattering profile. The read-out electronics consist of preamplifiers with pulse-shaping, which are mounted on the detector, and 12-bit, 20 MHz digitizers. The resolving time of the electronics is 300 ns, which is sufficient to isolate a single pulse of scattered X-rays when the synchrotron is operated with a hybrid or asymmetric fill pattern. The data acquisition hardware can average a programmable number of digital samples, up to 64, every 3.68 mus (the period of the synchrotron) to provides a single 12-bit average of the voltage from the analog amplifier chain. The temporal range of the detector is 3.68 seconds or longer and may be controlled by the experimenter. An alpha source is used to calibrate the detector and electronics, and document their performance. Preliminary results obtained during the commissioning of the detector are presented

A High-Speed One-Dimensional Detector for Time-Resolved Small-Angle X-Ray Scattering: Design and Characterization

A high-speed one-dimensional detector for time-resolved small-angle x-ray scattering has been designed and built for experiments at the Advanced Photon Source of Argonne National Laboratory. This detector is made from a 500-μm thick by 150-mm diameter ultra-high-purity n-type silicon wafer. The electrodes, which are a series of concentric rings that are deposited in the wafer, integrate the scattered x-rays over the azimuthal angle and, thereby, produce a one-dimensional detector. This design yields 128 rings, which allows parallel processing of the signal from each ring. The readout electronics consist of transimpedance front-end amplifiers, one for each ring, followed by active pulse-shaping filters. The amplifier signals are digitized using 12-bit analog-to-digital converters, one per ring, which operate at 20 MHz. The frame rate of the system is 271 kHz. Up to 220 - 1 scattering profiles may be stored on a random access memory chip and transferred to a data file at a rate of 16 × 103 profiles/sec. For X-ray energies between 3.5 and 13.2 keV the efficiency exceeds 80%. The resolving time of the electronics is 300 ns, which is sufficient to isolate electronically a single pulse of scattered x-rays when the synchrotron is operated in a hybrid or asymmetric fill pattern. Therefore, laser-pump/x-ray-probe experiments can be performed without a mechanical shutter. Examples of time-resolved speckle and the kinetics of the formation of sodium chloride particles are presented. This detector is capable of acquiring small-angle x-ray scattering profiles over multiple time scales, which are needed to characterize many chemical, physical, and biological processes. In addition, this detector may be tested and calibrated before experimental runs, without access to an intense beam of x-rays, with alpha particles from a radioactive source such as 241Am.

In situ X-ray data collection and structure phasing of protein crystals at Structural Biology Center 19-ID

A prototype of a 96-well plate scanner for in situ data collection has been developed at the Structural Biology Center (SBC) beamline 19-ID, located at the Advanced Photon Source, USA. The applicability of this instrument for protein crystal diffraction screening and data collection at ambient temperature has been demonstrated. Several different protein crystals, including selenium-labeled, were used for data collection and successful SAD phasing. Without the common procedure of crystal handling and subsequent cryo-cooling for data collection at T = 100 K, crystals in a crystallization buffer show remarkably low mosaicity (<0.1°) until deterioration by radiation damage occurs. Data presented here show that cryo-cooling can cause some unexpected structural changes. Based on the results of this study, the integration of the plate scanner into the 19-ID end-station with automated controls is being prepared. With improvement of hardware and software, in situ data collection will become available for the SBC user program including remote access.

Development of a real-time timing-shutter performance monitor for protein crystallography.

In order to accurately monitor shutter timing events and long-term shutter performance, a timing-shutter monitor has been developed. This monitor uses a photodiode to capture X-ray-induced fluorescence from the shutter blade in synchrony with goniometer rotation to measure shutter opening and closing delay times, as well as the total time that X-rays are exposed to the sample during crystallographic data frames.

Kodak CCD-based detector for small angle X-ray scattering

The Beamline Technical Support Group (BTS) at the Advanced Photon Source (APS) has developed two CCD detector systems (the Single and Quad Platinum systems), for x-ray diffraction and imaging experiments. Both of these systems, optimized for sensitivity toward x-ray photons, utilize the Kodak KAF-4320E CCD coupled to fiber-optic tapers, custom mechanical hardware, electronics, and software systems developed at the APS. Each CCD is composed of ~ 2k × 2k 25-μm-sized pixels, providing a total active detector area of 168 mm × 168 mm. In fast mode with 4 × 4 binning, the Quad system can reach a rate of 5 frames per second (fps). The sensitivity of the detector has been enhanced by using tapers with low demagnification ratio (1.78) and CCDs with high quantum efficiency. The sensitivity, resolution, and dynamic range have made the detectors suitable for a wide range of synchrotron experiment, in particular, small-angle x-ray scattering (SAXS). SAXS data from the x-ray scattering of silver behenate powder have been used to demonstrate the performance of the detector.

Biological crystal alignment using image processing

Crystal location and alignment to the x-ray beam is an enabling technology necessary for automation of the macromolecular crystallography at synchrotron beamlines. In a process of crystal structure determination, a small size x-ray synchrotron beam with FWHM as small as 70 μm (bending magnet beamlines) and 20 μm (undulator beamlines) is focused at or downstream of the crystal sample. Protein crystals used in structure determination become smaller and approach 50 μm or less, and need to be precisely placed in the focused x-ray beam. At the Structural Biology Center the crystals are mounted on a goniostat, allowing precise crystal xyz positioning and rotations. One low and two high magnification cameras integrated into synchrotron beamline permit imaging of the crystal mounted on a goniostat. The crystals are held near liquid nitrogen temperatures using cryostream to control secondary radiation damage. Image processing techniques are used for automatic and precise placing of protein crystals in synchrotron beam. Here we are discussing automatic crystal centering process considered for Structure Biology Center utilizing several image processing techniques.