The Macromolecular Structure Facility (MSF)

Introduction

The post-genomic era has ushered in the new field of structural genomics that links the advances in genomics and proteomics research (for example, the sequencing of entire genomes and the use of protein chips) with structural biology. The use of automation to clone, express, purify, and crystallize (Figure 1) wild-type and mutant proteins in tandem has led to a realization and advancement of structural genomics and high-throughput structural biology (Scientific American, April 2002). The number and size of structures determined by x-ray diffraction has steadily increased in the past decade, and the identification of key structural elements and details in the active site has enabled the assignment of the function of the protein target. The enormous developments that have occurred in biochemistry, microbiology, and molecular and structural biology continue to advance pharmaceutical and biomedical research. UIC has recognized that structural biology is a vital component of all current biomedical research, and that it will play an increasingly important role in the post-genomic era. With this in mind, it established the Center for Pharmaceutical Biotechnology and, more recently, the Center for Structural Biology. It is the Research Resources Center’s intention to support this cutting-edge research, and a state-of-the-art facility for macromolecular x-ray diffraction was established in 1998. We focus herein on a description of the field, the RRC Macromolecular Structure Facility (MSF), and the research carried out by a core set of campus researchers.

The Beginning of the MSF

In 1998, the MSF was founded with a relocation of the x-ray diffraction instrumentation from Dr. Karl Volz’s lab. Dr. Volz is a senior crystallographer in the Department of Microbiology and Immunology, and was the only protein crystallographer on campus for several years. With the completion of the Molecular Biology Research Building (MBRB), with the support of the Departments of Biological Sciences and Infectious Diseases and Immunology, the RRC moved the instrumentation to the first floor in the central core of MBRB. Dr. Volz moved his laboratory into new quarters across the hall from the MSF, and has served as director of the facility.

As part of an expansion into the area of structural biology due to a major UIC initiative, three new crystallographers were recruited to join the faculty, and were assigned new laboratory space in MBRB in 1999. Dr. Andy Mesecar joined the Department of Medicinal Chemistry and Pharmacognosy, and the Center for Pharmaceutical Biotechnology, in the College of Pharmacy; Dr. Arnon Lavie joined the Department of Biochemistry and Molecular Biology in the College of Medicine; and Dr. Connie Jeffery joined the Department of Biological Sciences and the Laboratory for Molecular Biology in the College of Liberal Arts and Sciences. In 2000, Dr. Bernie Santarsiero joined the Center for Pharmaceutical Biotechnology, and helps in the maintenance and operation of the MSF. He will also serve as director of the new facility for Small Molecule Diffraction (SDF, the Structure Determination Facility).

X-ray Diffraction and Crystallography

H. and W. L. Bragg, a father and son research team in England, first noted the interaction between x-rays and single crystals in 1912, following observations from Laue and Ewald. They exposed large crystals of zinc sulphide, rock salt, and other minerals to x-rays and recorded images with photographic film. In their investigations, the positions of the source, sample, and film were fixed. They were able to interpret the photographic images in terms of the makeup of the crystal samples and assigned individual atom positions. This led to the discovery that x-ray diffraction would be useful to determine the structure of various materials, including elements, alloys, inorganic and organic compounds, minerals, and polymers that compose a small array of atoms, or macromolecules (see Figure 2), that are composed of substantially larger arrays of atoms, such as proteins, enzymes, ribozymes, and viruses.

X-Ray Diffraction Instrumentation in the MSF

There are three fundamental components in the x-ray diffraction experiment: the x-ray source, the crystalline sample, and the detector.

The x-rays are generated by a source, most typically a sealed-tube, rotating anode (Figure 3), or synchrotron. The rotating anode is 2-10 times stronger than a sealed-tube source, and a synchrotron is 100-1000 times stronger than a rotating anode source. There are six synchrotron sources in the United States, and one site, the Advanced Photon Source (APS) is near Chicago at the Argonne National Laboratory. UIC is a member of two consortia, CARS (Corsortium of Advanced Radiation Sources, Sector 14) and SER-CAT (Southeastern Regional Collaborative Access Team, Sector 22). The synchrotron source offers a higher intensity and smaller photon beam size, enabling the collection of intensity data from extremely small and poorly diffracting crystals in a short amount of time. The rotating anode is the preferred choice for in-house laboratories since it offers high flux and can be easily maintained. Often samples can be screened in-house and saved, with a complete data set collected later at the synchrotron.

The sample, provided by the user, is typically a single crystal, roughly the size of a table salt or sugar crystal, or about 50-500 microns. Protein single crystals are very sensitive to a change in their environment, and the samples must be maintained in contact with the solution that they were grown from and sealed in capillaries or frozen in solution with some type of antifreeze (e.g., glycerol, polyethylene glycol, etc.).

There are several types of detector that are available for x-ray diffraction. The detectors are capable of converting an x-ray photon into an electrical current. Typically they have large apertures and relatively fast readout to collect the diffraction data most efficiently. Protein crystals do not diffract x-rays very well, so the detectors need to be very sensitive.

At present, there are two detectors available in the MSF for data collection: the Siemens Hi-Star Multi-wire area detector and the Rigaku/MSC R-AXIS-IIc area detector. A variable-temperature cooling system is in operation with the R-AXIS area detector, and can cool samples from -180 to 4C. A grant proposal to NIH/NCRR has been submitted to upgrade instrumentation in the facility next year.

The Siemens Hi-Star area detector is a photon counting detector, able to measure every x-ray photon with virtually no readout noise or dark current. The readout is in real-time, and a frame of data is stored almost instantaneously. An x-ray photon initiates an ionization event, and generates a charge and current detected at specific grid position.

The R-AXIS area detector has a large aperture, roughly 19cm square. It uses imaging plate technology in which an x-ray photon is stored as a color center, and then photo-converted to a light photon for readout with a photomultiplier tube. The result is an image with a large dynamic range, low readout noise, and no dark current.

The Experiment

The experiment is relatively simple. A single crystal is positioned in front of an x-ray source, and the resulting scattered array of x-rays is recorded. The x-ray beam is roughly 0.5mm in diameter, matching the size of the sample crystal. The majority of the x-rays will pass through the sample, but some will be deflected. Typically the crystal is rotated or oscillated in a small rotation angle, about 0.1 to 1^°, for several minutes. (At the synchrotron, the exposure time is for a few seconds). A typical image from the R-AXIS detector is shown in Figure 4. Then the crystal is rotated further and another image is recorded. Several images, from tens to hundreds, are recorded for analysis. For the analysis, the position and darkness of the “spot” is recorded. The darker the spot, the greater the number of x-ray photons that have been deflected to that position (Figure 4). The pixel size of the detector is 0.1mm, and the spots are several pixels in size. The processing is automated, and results in a list of spot positions and integrated intensities.

It is beyond the scope of this article to discuss how the atomic coordinates are derived from the x-ray diffraction intensity data. Depending upon the sample, a number of techniques have been developed over the past fifty years to derive the individual coordinates. Figure 5 shows one portion of a structure with individual atom coordinates in the active site engulfed by a net of “electron density.” In an iterative process, the atomic coordinates are adjusted to fit in the electron density. The resulting structure is a time-averaged, space-averaged, set of atom coordinates.

Once the atom coordinates are determined, a number of structural features can be assigned. These include the fold motif, assignment of the active site, hydrogen bonding, etc. Often active sites involve a metal ion (Figure 5).

Figure 4. An image recorded on the R-AXIS detector in the MSF by rotating the crystal. (hi-res image JPEG, 600x600, 24 KB)	Figure 5. Electron density maps of the active site of an enzyme that is involved in biodegradation of the industrial pollutant dinitrotoluene. The iron atom is clearly visible as is the enzyme inhibitor dinitrocatechol. The structure of this enzyme was solved by Bernie Santarsiero in the Mesecar laboratory.

We welcome collaboration with all campus researchers. Due to the complexity of the experiment and safety issues, potential users should contact any of the MSF users listed herein.

Research Highlights

The main purpose of the MSF is to facilitate the screening of samples and collect diffraction data from the best crystals. What follows are brief descriptions of current research interests from some of the major facility users (Figure 6).

Connie Jeffery (cjeffery@uic.edu) - Analysis of complete genomic sequences indicates that over 25% of an organism’s proteins are embedded in cellular membranes. Transmembrane proteins play vital roles in cell-cell communications, transmembrane signaling, ion transport, and maintenance of cell structure, and are the targets for the majority of pharmaceuticals in use today. In addition, the misfolding of specific transmembrane proteins results in diseases like cystic fibrosis, one of the most common lethal genetic diseases. The three-dimensional structures of transmembrane proteins are important for determining their mechanisms of function, and would aid in the development of new and better drugs for hypertension, depression, arthritis, cancer, cystic fibrosis, diabetes, and many other diseases. A computer-based analysis of known transmembrane protein crystal structures is being used to identify characteristics important in determining transmembrane helix packing. My lab uses a combination of methods, including x-ray crystallography, molecular biology, computer-based protein structure analysis, and biochemical characterization, to study the structure and function of transmembrane proteins.

Arnon Lavie (lavie@uic.edu) - Our laboratory focuses on enzymes that are important for the activation of prodrugs. Many medications are administered as inactive compounds, called prodrugs, and these molecules require some sort of metabolism, usually catalyzed by human cellular enzymes, to be converted into their active (i.e., therapeutic) form. Due to this reliance on enzymes for activation, and to the fact that many prodrugs are poor substrates of their corresponding activating enzyme, the maximum potential of these medications is often not achieved. The poor activation of prodrugs not only results in low concentration of the active metabolite of the drug but can also cause toxicity stemming from partially-activated metabolites. Therefore, understanding--on the molecular level--the determinants that limit the activation of various prodrugs is significant information that can be utilized in the development of more effective medications. The work conducted in the laboratory focuses on understanding factors that limit the activation of nucleoside analog prodrugs (e.g., AZT for AIDS and AraC for cancer).

Andy Mesecar (mesecar@uic.edu) - One of the remaining and great unsolved questions in chemistry and biology from the twentieth century is the nature of enzyme catalysis-How do enzymes accelerate the rate of chemical reactions to over 10¹⁷-times faster than the corresponding uncatalyzed reactions, and how do they achieve such high degrees of substrate specificity? The central hypothesis of the proposed research is that enzymes achieve these remarkable properties, not by some unconventional enzy “magical” property, but by combining general physical-chemical and structural properties with dynamic processes. We are integrating a variety of experimental approaches including structural biology, molecular genetics, and computational chemistry in order to explore the relationships between enzyme-dynamics and catalytic power and specificity of enzymes involved in disease and biological detoxification. Our principle tool for structure and dynamics studies is x-ray diffraction. We solve a number of structures in-house with data from the MSF, and also carry out elaborate time-resolved Laue x-ray diffraction studies at the Advanced Photon Source . The Laue method uses intense polychromatic x-rays in short (microseconds to picoseconds) bursts to capture a time-series of x-ray photographs that can be used to reconstruct a “3-D-molecular movie” of macromolecular motion at atomic resolution. These types of studies will allow us to see biological function in real-time motion.

Karl Volz (kvolz@uic.edu) - My laboratory uses the tools of protein crystallography to analyze the structural determinants of protein function in a number of different cellular processes. Our current research projects focus on the structure and function of a variety of serpins (serine-protease-inhibitors) in order to understand their roles in both diseased and healthy states.

In our first serpin project, we solved the structure of the anti-apoptotic viral serpin cytokine response modifier protein A (crmA).

More recently we determined the structure of the human non-inhibitory serpin known as pigment epithelium-derived factor (PEDF), a potent negative growth factor found in the retinal pigment epithelium of the eye (Figure 7). In healthy adult tissue, new vascular growth is suppressed, while in diseased states like retinopathy and cancer, vascular growth can go unchecked. PEDF is the major anti-angiogenic factor in the eye. The PEDF structure will enable us to design and construct peptide mimics for the control of diabetic retinopathy and tumor-associated angiogenesis.

These projects are carried out in collaboration with UIC investigators Drs. P. G. W. Gettins, S. T. Olson and P. A. Patston.

Bernie Santarsiero (bds@uic.edu) - My interests focus on the development and use of x-ray diffraction for structure elucidation, the detailed characterization of molecules at the atomic level, and as a tool to investigate mechanistic landscape of chemical reactivity. We have extensively used small molecule x-ray diffraction to assign novel connectivity and bonding properties, variable-temperature and low-temperature experiments to characterize potential energy surfaces, neutron diffraction to investigate hydrogen bonding patterns, polychromatic and time-resolved methodologies to trap transient states, and crystal engineering to develop new materials. Most recently, I directed a team of scientists and engineers at Lawrence Berkeley Lab and the Genomics Institute of the Novartis Research Foundation to develop automation for the high-throughput cloning, expression, purification, and microcrystallization of proteins. This project included the design and construction of beamline 5.0.3 at the Advanced Light Source with automation for sample manipulation, crystal centering, and data collection. Syrrx, one of two major structural genomics companies, was founded using this technology, and is a major contributor in the NIH/NIGMS Protein Structure Initiative. This technology promises to accelerate the use of the structure-based paradigm for pharmaceutical drug design.

News and Updates

Electron Microscopy Service (EMS)

Following meetings with Professor Robert Folberg, Head, Department of Pathology, and Dr. Gregorio Chejfec, Director of Anatomic Pathology, the EMS has taken over responsibility for diagnostic TEM specimen preparation and evaluation for the University of Illinois Hospital, using the JEOL JEM-1220. Some equipment from the Pathology Specimen Preparation lab has also been moved to EMS to facilitate carrying out this work. This includes a Reichart Ultracut E ultramicrotome that has replaced the Sorval MT6000, which was no longer fully operational.

Nuclear Magnetic Resonance Laboratory (NMRL)

The Nuclear Magnetic Resonance Laboratory (NMRL) is pleased to announce that the upgrade for our 360 MHz NMR spectrometer is complete. The 360MHz NMR spectrometer now has a 2-channel Bruker AVANCE console It is equipped with a 5mm QNP probe. The following four nuclei can be observed with this probe H1, C13, F19 and P31.

The OLISS reservation system is used with this NMR so that users can reserve time on the instrument after they have received training in its use. The upgrade was purchased so that the NMR community at UIC would have greater access to a modern NMR for routine analysis. This 360 NMR frees up time on the research grade 500 MHz NMR that is also in the laboratory.

DNA Sequencing Facility (DNAS)

In April, DNAS acquired a ABI 3100 Capillary Sequencer. After completing training and parallel testing of it and the gel-based system we previously used, we believe that our users will be happy with the results. Runtime on the capillary sequencer is 2.5 hours whereas the gel-based system required 10 hours.

In July, DNAS will institute a ‘same day’ sequencing service. Users who submit samples in the morning will have their results in the late afternoon. This service has been requested by many of our users but was not feasible until the capillary system was up and running.

Our Sigma-Genosys online oligo service has been a great success. In July, Operon will also be offering online ordering.

Our ABI Freezer program has been expanded to include items for Real Time PCR. Stop by the facility (3260 MBRB) to receive an updated list of available items.

Welcome Aboard

We are pleased to welcome Dr. William Hendrickson, Associate Professor of Microbiology and Immunology, to the RRC staff with a half-time commitment as an Assistant to the Director. He will be responsible for providing academic oversight for the DNA Sequencing Facility. In addition to his responsibilities in this key service facility, he will be evaluating the organization and function of the units that make up the biotechnology division of the RRC. In the past few years the RRC has experienced tremendous growth in this area. Bill will be able to devote more time to representing these units. In the process we hope to optimize, not only the operational efficiency of each unit, but truly integrate the biotechnology units in order to provide the best possible service to the users with the resources we have available.

Comings and Goings

A lot of moving of equipment and laboratories is on the horizon. On the west campus, a project is being initiated to prepare above-ground laboratories in College of Medicine, West (CMW) to house the wet chemical services of the Protein Research Laboratory. A major infrastructure upgrade and renovation is in the early development stages for the basement of the Medical Sciences Building (MSB). Demolition is expected to start in late summer or early fall. The Flow Cytometers will move into new laboratories as part of this project. Some disruption can be expected as the demolition and subsequent construction proceeds, but every effort will be made to keep equipment running.

On the east campus, preparations are in progress to prepare a laboratory to receive the new JEOL GCMate mass spectrometer. The mass spectrometry lab will move into new quarters with better temperature control. Parts for the new pulsed EPR (electron paramagnetic resonance spectrometer) have started to arrive, and final arrangements are being made to prepare the laboratory for installation of the various components as they are fabricated. This project is headed by Professor W. Andreas Schroeder (UIC, Physics).

Administrative Support

To streamline the process and associated costs of providing paper copies of the monthly billing vouchers to department business managers, we are investigating electronic distribution of the same information. Some departments will be asked to participate as we develop the new distribution format.