The Blu-Ice and Distributed Control System (DCS) software packages were developed to provide unified control over the disparate hardware resources available at a macromolecular crystallography beamline. Blu-Ice is a user interface that provides scientific experimenters and beamline support staff with intuitive graphical tools for collecting diffraction data and configuring beamlines for experiments. Blu-Ice communicates with the hardware at a beamline via DCS, an instrument-control and data-acquisition package designed to integrate hardware resources in a highly heterogeneous networked computing environment. Together, Blu-Ice and DCS provide a flexible platform for increasing the ease of use, the level of automation and the remote accessibility of beamlines. Blu-Ice and DCS are currently installed on four Stanford Synchrotron Radiation Laboratory crystallographic beamlines and are being implemented at sister light sources.

A three-dimensional crystal structure of the biotin-binding core of streptavidin has been determined at 3.1i-resolution. The structure was analyzed from diffraction data measured at three wavelengths from a single crystal of the selenobiotinyl complex with streptavidin. Streptavidin is a tetramer with subunits arrayed in D2 symmetry. Each protomer is an 8-stranded fl-barrel with simple up-down topology.Biotin molecules are bound at one end of each barrel. This study demonstrates the effectiveness of multiwavelength anomalous diffraction (MAD) procedures for macromolecular crystallography and provides a basis for detailed study of biotinavidin interactions.Streptavidin takes its name from the bacterial source of the protein, Streptomyces avidinii, and from hen egg-white avidin with which it shares an extraordinary ligand binding affinity (Kd -10-15M) for biotin (1). This similarity extends to many other properties (2), including a common tetrameric structure and a 33% identity in amino acid sequence between avidin and the homologous core of streptavidin (3, 4). Core streptavidin is proteolyzed naturally, but not always completely (3), at both ends of the 159-residue gene product to a 125-to 127-residue core (4) that matches quite precisely with the actual secreted avidin gene product (5). The biological functions of avidin and streptavidin are poorly understood, but they most probably involve antibiotic properties. Interest in the avidin family, however, transcends their natural biology. Their remarkable avidity for biotin motivates two types of study: (i) efforts to understand the chemical basis for the high affinity and (ii) attempts to optimize biotechnology applications that exploit this activity (6)(7)(8). We aim to examine these biophysical and biotechnological properties in refined crystallographic detail. Streptavidin has also been crystallized by others (ref. 9 and P. McLaughlin, personal communication).This structural study of streptavidin also has a second objective related to diffraction methodology. It seemed from the outset that selenobiotinyl streptavidin could be an apt subject for direct analysis from multiwavelength anomalous diffraction (MAD) data obtained with synchrotron radiation. Selenobiotin is a stable compound (10) sufficiently similar to biotin itself that the two molecules crystallize isomorphously (11). The high affinity (Kd 10-13 M) of avidin for desthiobiotin (2, 12) suggested to us that selenobiotin would also bind well. We expected anomalous diffraction ratios (13) from the four selenium atoms in the 54-kDa core streptavidin tetramer (up to 3%) that compared favorably with signals measured successfully from crambin (14) and myohemerythrin (15) and with those obtained in our lamprey hemoglobin test of MAD phasing (16,17).The theoretical basis for the MAD method and details of its implementation are presented elsewhere (13,17,18). Qualitatively, MAD experiments can be thought of as in situ multiple isomorphous replacements (MIR) generated by the variation in scattering fact...

An automated system for mounting and dismounting pre-frozen crystals has been implemented at the Stanford Synchrotron Radiation Laboratory (SSRL). It is based on a small industrial robot and compact cylindrical cassettes, each holding up to 96 crystals mounted on Hampton Research sample pins. For easy shipping and storage, the cassette ®ts inside several popular dry-shippers and long-term storage Dewars. A dispensing Dewar holds up to three cassettes in liquid nitrogen adjacent to the beamline goniometer. The robot uses a permanent magnet tool to extract samples from, and insert samples into a cassette, and a cryo-tong tool to transfer them to and from the beamline goniometer. The system is simple, with few moving parts, reliable in operation and convenient to use.

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