1997
The Structural Biology Synchrotron Users Organization (BioSync) was formed in 1990 to promote access to synchrotron radiation for scientists whose primary research is in the field of structural biology. The synchrotron radiation techniques used for structural biology can be sub-divided into four principal areas: crystallography, spectroscopy, scattering from noncrystalline materials and imaging. The BioSync membership includes leaders of all such structural biology research groups in North America.
The BioSync group had two organizational meetings and convened a Study Group in 1989/90. The main result of that activity was publication of a report in 1991 on the status of structural biology research using synchrotron radiation. It included the results of surveys of both the synchrotron radiation facilities and the structural biology research community. The Study Group of experienced structural biologists and synchrotron radiation experts met to evaluate the survey data, to assess the size and needs of the community, to predict what synchrotron radiation facilities would be needed in the future, and to write the report, which was published in July, 1991.
The main conclusions of the report were that structural biology, especially crystallography, was a very rapidly growing field with a growing impact on basic and applied biology, and that the synchrotron radiation facilities available at the time were insufficient for the needs of this ever-expanding community. Both construction of additional beamlines and improved support for existing beamlines were recommended to meet the predicted need. Construction costs were estimated at $3-4M per beamline and operation costs at $0.5-1.0M per year per beamline (1990 dollars). In its most controversial conclusion, the Study Group predicted a very large "latent" demand for synchrotron beam time from biologists who were not specialists in structural methodologies. More streamlined structural experiments coupled with an intense demand for new macromolecular structures were the driving forces for the "latent" demand.
This document is intended to provide a report on the current status of the biological uses and demands of synchrotron radiation in the U.S. and is an update to the 1991 BioSync Report. The synchrotron radiation facilities and user community have once again been surveyed, and a group of experienced structural biologists has analyzed the data and written this report. The 1997 BioSync Committee met in Cambridge, MA, in July, 1997, to analyze the results of the user and facility surveys, to consult with users and with representatives of funding agencies and to reach the main conclusions presented in this report.
The 1997 BioSync Committee members, and authors of this Report, are:
Others attending the meeting in Cambridge, MA, in July 1997, were:
The survey of synchrotron facilities was conducted by R. M. Sweet, NSLS. Electronic support for the user survey and data analysis was administered by Sheryl Martin and Laura Yust of the Human Genome Management Information System at Oak Ridge National Laboratory.
The meeting of the BioSync Committee was supported by the National Center for Research Resources, NIH, and the printing of this document by Office of Biological and Environmental Research, DOE.
Technical editting and layout of the report were by Janet Hollister, Dept. of Biological Sciences, Purdue University, cover design by Dr. Peter Kuhn, SSRL, Stanford University, and printing arrangement by Marjorie St. Pierre, SSRL. Printed copies of the report are available from J. Hollister, Dept. of Biological Sciences, Purdue University, West Lafayette, IN 47907 USA, and an electronic version is available at http://www.ornl.gov/hgmis/biosync/.
Janet Smith
December, 1997
In the six years since the last report of the Structural Biology Synchrotron Users Organization (BioSync), the impact of structural biology in all areas of biological science has expanded greatly, as anticipated. With this continued development has come an increase in both the size and the complexity of the macromolecular structures that are being determined and in the difficulty of the experiments that are being pursued. This increase in complexity, which was not expected to occur so quickly, has meant that synchrotron-based structural biology has expanded its role and now makes a significant contribution in addressing the fundamental questions of how life processes are carried out and the practical applications of treating disease at the molecular level. Thus, it is not simply that the number of macromolecular crystal structures is growing, but more importantly, that structural biology is having an increasing impact on such diverse fields as immunology, neurobiology, cell biology, virology, physiology, molecular biology, medicine and biotechnology.
These recent advances can be attributed to three key improvements in methodology: (1) the ability to clone and express a vast array of cellular proteins in quantities sufficient for structural studies, (2) the use of cryo-crystallography to prepare extremely stable crystals, and (3) the availability of and technological innovations at synchrotron radiation facilities. Many more macromolecules are now being crystallized. With the use of frozen crystals, crystallographic studies of large multicomponent complexes have now become nearly routine. High quality data are also being obtained from poorly ordered or weakly diffracting (as opposed to merely small) crystals. Non-crystallographic synchrotron techniques are also providing complementary information on systems with inherent conformational flexibility or with metal centers. These factors have brought many more projects of high biological significance into the realm of structural biology. Without synchrotron sources, many of these new research projects, which are often extremely challenging biophysical studies, could not yet have been undertaken. It is likely that the advances seen in the last six years represent only the beginning of an even greater explosion in the structural biology field that will accompany the on-going genome projects.
The BioSync Committee has evaluated the present synchrotron needs of the structural biology community through surveys of both the community and the synchrotron facilities themselves and through meetings with experienced users as well as representatives of government funding agencies. The object of this report is to evaluate what synchrotron facilities and support operations are currently needed, and to anticipate what will be required to sustain the exciting progress in structural biology in the coming years. The following main conclusions have been reached.
1. Structural biology research is producing results of high biological impact that have a direct bearing on human health issues.
The 1991 predictions have been borne out and in many ways surpassed. Structure-based drug design, which seemed merely a trendy phrase a few years ago, has become a reality. The design of new medically important drugs, such as the HIV-protease inhibitors and the influenza neuraminidase inhibitors, is a direct consequence of research in structural biology. It is expected that this trend will continue and will become increasingly important in the fight against the plethora of emerging and re-emerging viral and microbial pathogens that are now infecting the human population. Currently, no drugs are available for protozoan diseases such as sleeping sickness, malaria and Chagas' disease. New therapeutics are also required to combat drug-resistant pathogens, such as some forms of tuberculosis, which are no longer controlled by currently available drugs. Structural biology is also becoming increasingly important in biotechnology, as for example, in the design (or re-design) of enzymes to degrade pollutants or to act as thermostable industrial catalysts, or in the design of insecticides with increased efficacy. These biotechnology applications can have huge environmental and economic impacts. In the basic sciences, the fields of cell and developmental biology have become "molecular" through over-production of extremely interesting macromolecules in sufficient quantity for structural study. Many of these molecules are structurally challenging due to their size and complexity. On the horizon, major new insights are expected into the processes of cell biology and development that will come from the structures of key macromolecules, akin to the revolution in molecular immunology caused by the structure determination of the major histocompatibility antigen (MHC) class I molecule.
2. Synchrotron radiation is now a dominant contributor to new macromolecular structures.
The role of synchrotron radiation in structural biology has been growing rapidly over the last twenty years. A recent survey of the literature shows that synchrotron radiation was used in nearly half of the new structure determinations. The benefits of synchrotron radiation include substantial increases in resolution over those available with laboratory sources and the ability to study crystals that are too small or have a unit cell too large to be studied using home X-ray sources. Time-resolved studies by Laue methods have generated snapshots of enzyme reactions, and dynamic structures in solution have been investigated by small-angle scattering.
3. Synchrotron radiation combined with MAD phasing has revolutionized macromolecular structure determination.
Since 1991, the greatest technological advance in structure determination has been the full development of multiwavelength anomalous diffraction (MAD) phasing methods. This technique has the advantage of accurate and rapid structure determination using diffraction data from one crystal, once an appropriate chemical element has been incorporated in the macromolecule. Thus, MAD bypasses the rate-limiting step of finding isomorphous derivatives. The power of MAD is greatly enhanced by the widespread applicability of the selenomethionine label in proteins and the brominated uracil label in nucleic acids. A disproportionately large number of structures solved with MAD are now being reported in Science, Nature and Cell, attesting to its importance in structural problems of broad biological significance. MAD, which has an absolute requirement for tunable synchrotron radiation, will continue to have a major impact on the practice of structural biology. It is being adopted so rapidly that adequate MAD beam time is expected to be the most limiting synchrotron resource for structural biology in the next five years.
4. Non-crystallographic applications to structural biology continue to grow.
X-ray absorption spectroscopy, small-angle X-ray scattering and X-ray imaging continue to provide crucial information for systems that are dynamic, are very large or include metal centers. In addition, these methods complement crystallography in providing information critical for understanding biological function. For example, X-ray crystallography and X-ray absorption spectroscopy in combination provide a complete description of metal sites in proteins that is not provided by either technique alone. Metalloproteins are involved in all biological energy-capture, conversion and transfer. Small-angle scattering can be used to determine how protein components assemble into functional units and what changes in association are relevant to function. The recent construction of dedicated small-angle scattering beamlines at SSRL and APS has made this technique accessible to a much wider array of problems. Recent advances in X-ray microscopy and X-ray microprobe imaging offer the promise of dramatically improved images of cells and tissue.
5. The general demand for structural information in all molecular fields of biology continues to grow very rapidly, and is paralleled by a growth in the demand for synchrotron time.
Three factors contribute to the substantially increased demand.
a. Technological improvements in synchrotron facilities, X-ray detectors and crystal handling have brought many more biological problems into the range of structural biology and have significantly improved the success rate and quality of synchrotron experiments. The ability to freeze macromolecular crystals at cryogenic temperatures (ca. -170° C) has effectively immortalized many crystals. No longer is radiation damage of the specimen a major concern that limits the ability to measure high-quality data. More accurate, higher resolution structures have resulted from complete data sets being obtained from a single frozen crystal. The freezing process has also allowed use of smaller crystals, which require more irradiation in order to measure their weaker diffraction. Crystals with dimensions of less than 0.1 mm now routinely furnish high quality data. The combination of smaller crystals and harder X-rays has minimized absorption errors; and the low background characteristic of good synchrotron beamlines makes it easier to record excellent data at very high resolution. Hence, structures that were previously inaccessible have become almost routine. These advances coupled with MAD phasing have made high resolution structures attainable more rapidly from a greater proportion of crystals for a wider range of biological problems.
b. As the complexity of the biological project increases, there is greater demand for synchrotron time to tackle more difficult problems. These projects include crystals with very large unit cells, poorly scattering crystals of macromolecular assemblies or membrane proteins, micro-crystals, and problems in dynamics where the goal is to capture snapshots of biological events along an enzyme reaction coordinate or other kinetic pathway. For such complex problems, the ability to integrate information from several types of experiments - crystallography, spectroscopy, solution scattering or imaging - is often critical.
c. As anticipated, a significant new demand has indeed come from a "latent" community of users who are not specialists in crystallography but who have biologically significant structure determinations to carry out. These types of problems include multiple mutant structures, drug or ligand complexes of solved structures, molecular replacement structures and entirely new structures. Latent demand is difficult to quantitate, but it is already clear that many non-specialist laboratories are embarking on structure determinations. Their willingness to undertake structural work is a testament to the impact of structural results in molecular fields of biology and to the success in streamlining structural experiments. Non-specialist users are a greater challenge for synchrotron facilities because they usually need a higher level of assistance from scientific staff. In addition, the synchrotron facility may be their only available X-ray source. Non-specialist demand is also expected to rise rapidly due to structure determinations arising from the genome projects. Thus, additional, highly trained support staff are required to meet the needs of larger numbers of non-specialist users who will come to the synchrotron facilities. The designation of the specialist and non-specialist researcher is gradually blurring as structural science becomes more accessible and the biological problems to which it is applied become more challenging. Postdoctoral associates and graduate students in specialist laboratories are becoming more sophisticated biologists but are often less expert in biophysics than when structural studies were a more arduous and labor-intensive undertaking. This means that in the future even synchrotron users from specialist laboratories will require a higher level of support at the synchrotron facility than is required now.
6. Regional facilities will grow in importance.
Without question, there is a strong demand for regional facilities that can provide service to the regional scientific community. There was overwhelming enthusiasm and uniform support among structural biologists for keeping all of the current synchrotron facilities in the U.S. open for biological use in order to service regional needs. It was deemed of extreme importance that research groups be within driving or short flying distance of synchrotron facilities to exploit their resources fully. The ability to drive to a local facility with samples in hand was rated as extremely important by a majority of users. Graduate students and postdoctoral fellows are the majority of scientific workers who actually go to the synchrotron, and it is essential that proximity to synchrotron facilities allows them to travel in large numbers for training. As actual costs of synchrotron trips are almost never covered completely by research grants, the decreased travel costs associated with regional facilities are especially important.
7. The most cost effective way to improve throughput at synchrotron facilities is to upgrade existing beamlines.
In the 1991 BioSync Report, it was strongly advised that multiple new beam-lines be built. Much of this increase has been realized, especially with the new APS facility at Argonne and new ALS facility at Berkeley. Additional beamlines for biological use have become available at NSLS (Brookhaven), at SSRL (Stanford) and at CHESS (Cornell). These capital investments are expensive but essential. Unfortunately, after the initial mega-investment, only limited funds are typically available to keep the beamlines current with technological advances and adequately staffed to support user research. Consequently, the X-ray detectors and computer technology on most beamlines lag behind the state-of-the-art. It is strongly encouraged that beamline instrumentation at national synchrotron facilities be upgraded approximately every three years. These upgrades could substantially improve the throughput, and hence effective beam time, with a relatively small investment of funds and without the need for construction of expensive new beamlines. Since the best hardware and software are ineffective if well-trained staff are not available to assist the users, these upgrades must be accompanied by appropriate levels of staffing for user support.
Also, the structural biology community has become more organized since 1991 in response to its substantially increased reliance on synchrotron radiation. A substantial investment has been made in new beamlines by consortia of users from both the academic and industrial communities, who have purchased exclusive rights to as much as 75% of available time on individual beamlines. This trend is likely to continue as groups wish to have greater control of synchrotron beam time allocation for more convenient and rapid scheduling. Many users noted that the wait for beam time is not well matched to either the pace of science or the lifetime of samples that cannot wait for months before being used. The ponderous peer review system currently in place for beam time allocation at most synchrotron facilities is usually not appropriate for research projects that have already been peer reviewed for their primary funding. Alternative mechanisms for quicker and easier access to beamlines should be promptly developed.
8. Increased cooperation between organizations funding synchrotron facilities and basic research is highly recommended.
At present, the DOE, NIH/NCRR and NSF provide major funding for the national synchrotron facilities, whereas the majority of academic users are funded by NIH Institutes other than NCRR, or by NSF. The light source operations are provided mainly by DOE (ALS, APS, NSLS, SSRL) and NSF (CHESS). NIH/NCRR and DOE/OBER provide support for operation of beamlines for structural biology. Collaboration of these organizations would increase operational efficiency and planning for synchrotron source upgrades and the infrastructure required to operate the synchrotron facilities at the state-of-the-art. The periodic upgrade of beamline instrumentation and user support could be more effectively coordinated through such a collaboration. Government and other organizations funding research grants to the structural biology user community could provide up-front support in addition to the modest travel allocations in individual research budgets.
In summary, the recommendations are that all current beamlines must be effectively maintained at National centers in Brookhaven, Cornell, Argonne, Stanford and Berkeley. Allocation of funds to upgrade existing beamlines is highly desirable in order to accommodate increased demand. Collaborative funding of these resources by NIH, DOE and NSF is strongly encouraged. While it is expected that demand will always outstrip available resources, it is the strong opinion of the BioSync Committee that the increased demand is not simply a matter of an increased number of users or of more projects of the same type. Extraordinary advances have been made recently in structural biology that impact all aspects of the medical and biological sciences. Breakthroughs in complex macromolecular structures have included the proteasome, GroEl, the nucleosome, the TCR-MHC complex and muscle proteins. The fundamental difference is a move from small soluble molecules of up to 100,000 daltons to multimeric complexes of greater than 1,000,000 daltons. The consequence of this shift towards increasingly complex structures and complex biological and biophysical problems is that it is now possible to address fundamental aspects of how a cell functions and how genes and gene products control cell development and function. Understanding of the mechanisms of these fundamental life processes can be harnessed in the future for gene therapy, in the design of new drugs for treatment of a large number of human diseases and inherited conditions, and in applications to improve the environment. Structural biology has moved from the simple study of structure and function of single proteins to a molecular understanding of cellular processes that control life and death.