Undulator Resource for Structural Biology

Undulator Resource for Structural Biology

Research Emphasis

The Undulator Resource for Structural Biology is a facility for macromolecular crystallography at Sectors 8 and 24 of the Advanced Photon Source (APS) at Argonne National Laboratory (ANL). The resource is overseen by the Northeastern Collaborative Access Team, which includes scientists from Columbia University, Cornell University, Harvard University, Massachusetts Institute of Technology, Memorial Sloan-Kettering Cancer Center, Rockefeller University, and Yale University. Research emphasis is placed on signal transduction, DNA transcription initiation and regulation, cell cycle regulation, virus structure and function, membrane proteins, protein folding, and enzyme structure and function. Many of the research projects focus on how biological molecules interact to form large macromolecular complexes. The macromolecules studied by resource users often involve large unit cells, small crystals, weakly diffracting crystals, and crystals with weak anomalous scattering. Construction of the undulator resource involves development of a novel dual undulator design utilizing components developed by the APS. Other technologic research includes use of diamond monochromators, focusing optics, methods of phase determination, radiation damage, X-ray detectors, automated sample mounting, microdiffraction, and crystallographic software.

Current Research

Our primary technologic research involves designing and constructing a third undulator beamline, continuing to optimize the beamlines for experiments with challenging samples, adding microdiffraction capabilities to our beamlines, relocating our bending magnet beamline at Sector 8 (with its automated sample mounting system) to Sector 24, and continuing to develop software for crystallographic computing. The core science emphasis is to solve new, technically challenging crystal structures. These include macromolecular assemblies and membrane proteins for which the crystals are frequently small and weakly diffracting with large unit cells. In many cases, MAD phasing will require high-energy resolution because of weak anomalous signals or the presence of many anomalous scattering atoms.

Resource Capabilities

The Undulator Resource for Structural Biology provides advanced synchrotron beamlines for macromolecular crystallography optimized for use in MAD and SAD phasing. The resource also provides laboratory space for protein production and crystallization and office space for data analysis. The undulator resource currently provides a high-brilliance undulator beamline and one bending magnet beamline. An additional undulator beamline is nearing completion and a fourth is in the planning stages. Each beamline is equipped with X-ray detectors, cryocoolers, and other instrumentation for X-ray diffraction.

Instruments

8-BM (end-station): Open to general users. Source is a bending magnet. Station features include a precollimator mirror, vertical offset, Kohzu HLD8-24 dual-crystal focusing monochromator (6.5 -14 KeV, 2 eV bandpass), monochromator cryopump, P4-50 shutter, collimator assembly, Integrated Kirkpatrick-Baez focusing system, ADSC Quantum 315 detector, Oxford Cryojet, Advanced Light Source automated sample mounting system. The components of this beamline will be relocated to sector 24.

24-ID-C (end-station): In the commissioning phase; will open to general users in October 2006. Features include a vertical offset diamond monochromator (8-13.5 KeV, 2 eV bandpass), Kirkpatrick-Baez focusing, target flux (10¹⁴ ph/s in 100 times 100 mm² at 12.66 KeV), focal spot size variable down to 50 µm horizonal X 20 µm vertically, precision crystallographic goniometers, fast monochromatic rotary beam shutters, "click-n-center" crystal centering, Oxford Cryojet cryocooler, FTS Air-Jet crystal cooler. (-40 to +100c), pneumatically actuated beam attenuators, and split diode beam position monitors, ionization and sample fluorescence monitors, precision, remotely adjustable miniature beam stops, and video imaging microscopes.

24-ID-E (side-station): In construction; open for commissioning in late fall 2006. Features include a single crystal, side bounce horizontal offset diamond transmission monochromator (fixed energy of 12.66 KeV or 14.84 keV, 2 eV bandpass), Kirkpatrick-Baez focusing, and target flux (10¹⁴ ph/s in 100 times 100 mm² at 12.66 KeV). An MD2 microdiffractometer will be installed in fall 2006.

24-ID-D (fixed-wavelength side-station): Planning stage. Features include a diamond transmission, inboard-projecting undulator with horizontal offset monochromator (8.5 - 17 keV, 2 eV bandpass), Kirkpatrick-B¹⁴ X-rays/mm²/sec.

Other facilities: 8,000 square feet of laboratory and office space in LOM436, including biochemical and computing facilities. Chemical laboratory equipped for protein production and purification, including clean electrical power, Millipore filtered high-purity water, mandated safety equipment, standard chemicals, analytical balances, pH meters, an ultrasound dismembrator, a table-top refrigerated shaker, a high-speed refrigerated centrifuge, a UV/V spectrophotometer, stereozoom microscopes, Agilent 2100 Bioanalyzer, dynamic light scattering system, two incubators for crystallization (4 °C and 18 °C), -35 °C refrigerator for storing proteins, and an AKTA/fast protein liquid chromatography system for high-throughput automated analytical and preparative protein purification, 9- x 10-foot walk-in cold room equipped for chemistry and mounting of crystals.

Computers: MS personal computers-based computers for office functions and Linux-based personal computer systems for scientific and beam line applications at the APS site. Each staff member has his or her own computer, and the beam lines have computers available for users in addition to the extensive computer environment associated with the beam line controls, data acquisition, and data achieving. All computers are connected to high-speed hard-wired Local Area Networks with high-speed broadband access to the Internet. An APS-wide wireless system is readily available so that laptop computers with wireless capability can be carried and used anywhere within the APS complex. ANL also provides Virtual Private Network connections so that staff, with the necessary authorizations, can access the internal computer systems from off site. APS and ANL provide extensive firewall protection against hackers and spam e-mail.

Software

Current software includes HKL2000, SOLVE, Sistina, CNS, ccp4, Console, and the Blu-Ice graphic user interface. The scientific staff is exploring different software packages for fully automated structure solution that deal with all steps from diffraction image data processing to structure solution without customized scripts or format conversion. Automated suites under consideration include X-solve, ACrS, and HKL2000-PH.

Special Features

The first MD2 microdiffractometer at a synchrotron beamline in the United States will be installed in the early fall 2006; first user use is anticipated to be in late fall 2006.

Training Opportunities

Beamline users are given individualized training in safety, use of beamline software and hardware, and strategies for collecting optimized diffraction data. Staff scientists also provide instruction on the available crystallographic software for data analysis and structure solution.

Publications

1. Higgins, L. J., Yan, F., Liu, P., Liu, H. W., and Drennan, C. L., Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme. Nature6:838-844, 2005.

2. Rubin, S. M., Gall, A. L., Zheng, N., and Pavletich, N. P., Structure of the Rb C-terminal domain bound to E2F1-DP1: A mechanism for phosphorylation-induced E2F release. Cell 123:1093-1106, 2005.

3. van den Berg, B., Clemons, W. M. Jr., Collinson, I., et al., X-ray structure of a protein-conducting channel. Nature 427:36-44, 2004.

4. Xiong, Y. and Steitz, T. A., Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 430:640-645, 2004.