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--> 6 The Macromolecular Crystallography Resource at the Cornell High-Energy Synchrotron Source User Facilities for Protein Crystallography at Synchrotrons Synchrotron x-ray sources, used extensively for diffraction studies of biomolecular structures, are an example of a mature set of shared instruments and facilities. At the present time there are eight such x-ray sources worldwide with significant capabilities for structural biology research, and three additional installations will soon to be operational or under construction. Synchrotron radiation sources are expensive and can be constructed only through the cooperation of a large research community. Typical users of synchrotron radiation sources include physicists, materials scientists, biologists, chemists, and others. The larger community must support the construction of the synchrotron storage rings (which cost several hundred million dollars), while smaller groups of researchers band together in order to instrument individual beam lines for specific types of experiments. The cost of an individual beam line is several million dollars for construction plus ongoing operating costs. Individual scientists who use these resources share x-rays, instrumentation, and some software. These facilities are a rich source of information about what works in shared facilities and where there are problem areas or bottlenecks. The past decade has witnessed a dramatic increase in capabilities for determining the three-dimensional structures of macromolecules. New structures currently appear in high-impact journals such as Science, Nature, and Cell at rates approaching one per week and have profoundly affected every area of the biological sciences. Macromolecular structures produced by x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are used to understand the structural basis of protein function, resulting in applications to fields such as drug discovery and protein engineering. Synchrotron radiation
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--> sources, once considered a novelty, are now an essential tool for structural analysis. The MacCHESS Research Resource The Macromolecular Crystallography Resource at the Cornell High Energy Synchrotron Source (MacCHESS) provides support for the collection and analysis of x-ray diffraction data from crystals of biological macromolecules using synchrotron radiation. The overall goal of the MacCHESS research resource is to ensure that world class research in structural biology is performed at CHESS. This goal is accomplished by providing specialized equipment for macromolecular crystallography as well as trained support staff to assist outside users. The MacCHESS staff of two scientists, three technicians, a computer programmer, a machinist, and a secretary has established an active research program designed to advance the frontiers of synchrotron radiation research and structural biology. MacCHESS receives its major source of funding from the Biomedical Research Resource Program of the National Center for Research Resources at the National Institutes of Health (NIH). MacCHESS is dependent on the continued operation of the CHESS laboratory, which is responsible for delivering synchrotron radiation to the experimental hutch. CHESS is funded by the National Science Foundation (NSF) to provide synchrotron radiation for a wide variety of experiments. Structural biology accounts for about 35–40 percent of the total experiments performed at CHESS. CHESS, in turn, is dependent on the operation of the CESR (the Cornell Electron-Positron Storage Ring). CESR is maintained by the Laboratory for Nuclear Studies for use in particle physics experiments and is funded by the NSF. Core Research Projects Core research projects are performed by MacCHESS faculty and staff and are intended to advance the capabilities of the research resource. Core research projects provide the driving force for new developments such as advanced x-ray detectors, cryo-crystallography apparatus, new beam line optics, new x-ray instrumentation, and new data analysis software. Current core research projects include structural analysis of various targets for drug design, elucidation of enzyme mechanisms and protein engineering.
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--> Collaborative Research Projects Collaborative research projects are intended to extend new developments to a broader research community. Collaborators obtain early access to new instruments, techniques and methods and provide additional impetus for their development and refinement. For example, collaborative research was used as a mechanism for testing new x-ray detectors and the multiple wavelength anomolous diffraction (MAD) phasing instrumentation. User Research Projects (Service) Mature methods are made available to outside scientists using the facility on a competitive basis. In 1995, MacCHESS users performed experiments that included preliminary crystallographic analysis, high-resolution data collection, data collection for large unit cells, multiple isomorphous replacement (MIR) structure determination, molecular replacement structure and MAD phasing experiments. More than 200 scientists from 45 laboratories used CHESS facilities for macromolecular crystallography experiments during 1994. This work resulted in dozens of scientific publications and presentations at meetings. Training and Dissemination Workshops and Symposia As a user facility, MacCHESS provides visiting scientists with on-site training for all aspects of macromolecular crystallography including crystal freezing, experimental design, operation of station bench cameras, use of both image plate scanners and charge-coupled device (CCD)-based x-ray detectors, and evaluation and processing of data using various processing programs. Each year, CHESS organizes a users' meeting and workshop. The users' meeting features reports of research and development activities by both the local staff and outside users. The topic of the workshop relates to macromolecular crystallography about every other year. Training Videos MacCHESS has produced the first of a series of training videos on macromolecular crystallography using equipment provided by the Keck Laboratory for Molecular Structure at Cornell University. The first training
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--> video focused on cryo-crystallography and has been distributed to more than 400 scientists worldwide. Other videos covering various aspects of macromolecular crystallography and synchrotron radiation are planned. CHESS Newsletter Each year, CHESS publishes a newsletter that highlights the productivity and capabilities of the CHESS laboratory. In recent years, nearly half of the contributions have been in the area of structural biology. Future newsletters are planned to keep the community informed about ongoing and planned CHESS activities. MacCHESS World Wide Web (WWW) Home Page MacCHESS has established a WWW home page with which users can keep up with the latest developments in instrumentation, software, progress, and opportunities from MacCHESS. From a separate CHESS home page, users can learn about new CHESS developments and obtain beam time application forms. The WWW page has already proved to be an effective way for users to remain informed about MacCHESS in the time between CHESS newsletters. Ownership and Access Issues Synchrotron sources originally served primarily as research facilities for a select group of participating scientists who were developing the methodology and the technology. Now, however, there is an strongly increasing user demand for access to these facilities, particularly by the crystallography community. Access to CHESS by outside investigators is through competitive proposals based on the peer review process. For types of proposals are available: Program Proposals, Standard Proposals, Express Mode Proposals, and Feasibility Studies. Deadlines for Standard Proposals (requesting a block or blocks of time for a single experiment or structure, with approval good for two years) and Program Proposals (for a series of linked experiments or related structures over a four-year period) are announced about every six months. The proposals are sent to external reviewers for evaluation, and a final priority score is assigned by a proposal evaluation committee comprised of scientists representing all major areas of synchrotron radiation research. Access to CHESS is based on scheduling requirements and the final priority score.
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--> Express Mode Proposals were implemented to address the need of macromolecular crystallographers for rapid, short-term access to synchrotron radiation. A portion of the total beam time is set aside for these purposes based on the level of demand but is restricted to limit adverse effects on other types of proposals. Express Mode Proposals are normally limited to a maximum of 48 hours of beam time and should not involve hazardous materials. Express Mode Proposals are evaluated by a three-person committee, and beam time is allocated based on the committee's evaluation. Feasibility Studies are short-term access (up to four days) proposals that provide greater flexibility than Express Mode Proposals. The proposals are evaluated by a separate committee, and beam time is allocated based on the committee's evaluation. Feasibility Studies may involve hazardous materials and therefore may require approval from the Safety Committee. The procedure works satisfactorily for most users, and about 40–50 percent of the good projects gain access to synchrotron time. Access is currently reviewed independently of grant support for the projects involved, which potentially creates a chicken or egg dilemma. However, since synchrotron time is even more limited than grant support, the issue of awarding time to an unfunded project appears to be rarely, if ever, faced. Some of the most active scientists using synchrotron time are peripatetic wanderers who submit multiple applications to multiple facilities and use time wherever and whenever it can be found. To date, the system appears to have proved itself to be reasonably capable of dealing with this bit of redundancy. However, peer review of the same proposals for beam time by committees at several sites will increase significantly as the number of stations for protein crystallography studies in the United States approximately doubles in the next few years (up from the current 9 to about 20). Coordination of proposal review and scheduling among all facilities would be a significant logistical task, but facility directors should begin to explore mechanisms of coordination along with simplified review and scheduling algorithms that could reduce the administrative burden significantly. Rapidly increasing demand from nonspecialists (i.e., biologists or other scientists with an interesting molecule but no experience with a synchrotron or maybe even with crystallography) has underlined a problem facing all user facilities. There is a need to balance core research, which keeps the staff enthused, with collaborations and service to outside investigators. Despite the addition of two new beam lines, MacCHESS still has the same staff and budget as when it had only one, and a lengthy backlog of approved projects waiting for beam time has developed. The director is actively seeking new sources of funding for core research, in order to focus more of the existing staff's effort on user support. Ownership issues are perhaps less complex at MacCHESS than with some of the other case studies in this report. In the typical study, an investigator
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--> comes with his or her crystals and leaves with all the data. That said, there are also collaborations involving core staff and outside investigators, and there is no hard-and-fast rule about authorship at MacCHESS. The director's view is that purely technical assistance deserves an acknowledgment in any subsequent papers (and authors who overlook that are quickly notified), but that the basis of coauthorship negotiations should be contribution to the interpretation of data (as opposed to simply enabling data collection). Cost Issues As noted above, MacCHESS is supported by a grant from the National Center for Research Resources (NCRR) at NIH. That funding comes to approximately $1 million annually. This apparently straightforward arrangement is complicated by the fact that MacCHESS is dependent on CHESS, which has an annual budget of $2 million funded by NSF. CHESS in turn is dependent upon the operation of the half-mile-circumference CESR, which has annual operating costs of $15–20 million, also provided by NSF. Underutilization of the facilities because of insufficient funds to keep the synchrotron running throughout the year, or because of the competing needs of biological users and high-energy physics users, has been a significant problem for the other four U.S. synchrotron sources, all of which are operated by the Department of Energy (DOE). The DOE scientific facilities initiative of FY 1996 provided these facilities with an increase in operational funding to ensure full-time synchrotron operation. Cornell is the only synchrotron source funded by NSF and, thus has been affected only indirectly (changing demand for beam time) by these changes in DOE funding, but the dependence of the structural biology community upon support for a very high budget physics program is an obvious hazard in an era of tight money. At the level of MacCHESS, it is important to note that the basis of NCRR support is cutting-edge methodological research by core staff, rather than service to structural biologists from other institutions. This has not been a problem to date, but it does allow the possibility of success (in attracting users) putting a shared resource out of business. User fees generally are not assessed against the grants of outside investigators, although they are charged the costs of consumable supplies (x-ray film, etc.). Commercial users who insist that their work is proprietary are however charged for beam time (including the necessary services of staff) at the rate of about $800 per day. This charge seems unlikely to offset the full cost but may cover incremental costs. Proposals from industry are at somewhat of a disadvantage in the review process leading to beam time scheduling, because industry proposals generally deal with protein structures that are
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--> already known (e.g., human immunodeficiency virus (HIV) protease). At least at present, the prospect of breaking entirely new ground seems to weigh heavily in the review committee's decisions. Perhaps for this reason and because of the need to meet developmental timetables, a group called the Industrial Macromolecular Crystallography Association has raised enough money to build two beam lines at the advanced photon source about to open at Argonne National Laboratory. In return for ''their own'' beam lines, they have promised to give 25 percent of beam time to independent outside users. A similar arrangement might be a solution to both the handicaps faced by industry submissions in the review process at MacCHESS and the increasing demand for services of the staff by inexperienced users. Other Issues and Problems In the past, the effectiveness of synchrotron sources was often compromised for rather picayune or cost-ineffective reasons. Local infrastructure for biological experiments has often not been topflight at many synchrotron sources. This needs to be watched and corrected since the costs are often trivial compared with the cost of otherwise wasted synchrotron time. The current situation is reported to be largely satisfactory. Another area in which progress is needed is stronger local software support at the facilities. This will allow preliminary data analysis to be carried out almost in synchrony with the gathering of experimental data so that some problems can be caught early and corrected and the sheer volume of unprocessed data that has to be carried off-site and kept intact can be reduced to an acceptable level. A final problem that threatens to limit the utility of multiuser synchrotron sources is the requirement to travel to the site. Traveling to such remote facilities is an experience outside the culture of most biomedical or biological researchers. This will have to change as unique, expensive facilities become the norm in other areas such as very high resolution magnetic resonance imaging, accelerator mass spectrometry, and very large scale DNA sequencing to name just a few. A key issue for the design and operation of such facilities is whether the experimental users must come to the site or whether just their samples can be sent. Clearly, the more the latter mode of operation can be adopted, the less disruptive and the more effective shared resources will be. There seems to be no reason why, for the many routine types of applications, remote access will not suffice, and the sorts of remote monitoring that have become common in medical practice could easily be adopted to handle the vast majority of experimental situations. What must be accepted is the local cost of supporting significantly increased staffing at these facilities, whose role would be mainly—if not entirely—the support and service of external users. The
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--> danger in this model is possible stagnation in the continued development of novel capabilities for new science. Attention must be paid to a balance between these competing needs (a competition likely to be exacerbated in an era of restrained funding).
Representative terms from entire chapter: