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Synchrotrons and industrial structuralbiologyAndrew J. Howard

Pharmaceutical and chemical companies play an increasingly significant role in the use and development ofsynchrotron facilities for structural biology.

even collect complete data sets from aremote location. Web-based tools providea mechanism for both viewing images andremotely controlling the experimentassuming the sample has been installedand optically aligned by a local staff mem-ber. Although this would likely create anew set of issues for facilities, the longterm gains in user throughput could justi-fy the effort.

Full service crystallography. A furtherextension of remote access would be toprovide complete data collection andstructure determination services. Thiswould be particularly beneficial for non-specialists who have crystallized interest-ing and important proteins but lack theexpertise to carry out the subsequent stepsof analysis. The collection of data by tech-nical staff who are familiar with the localfacilities offers many potential advantagesin efficiency. However, in most cases, thefunding, which could approach one mil-lion dollars per beamline per year, is notcurrently available for synchrotron radia-tion facilities to provide this level of ser-vice. One possibility is for commercialenterprises to assemble full service capa-bilities on a for-profit basis. A drawback isthat projects might be prioritized on thebasis of financial rather than scientificpotential.

ConclusionIn summary, synchrotron radiation hasbecome a critical resource for structuralbiology. At the same time this resource islimited by demand from both structuralbiologists and scientists from other fields.Although significant progress has beenmade in recent years, we must continueefforts to make efficient use of existingfacilities through the development of newinstrumentation and new methods.Further gains in efficiency may beachieved by modifying the way in whichbeam time is allocated to individual usersand by exploring new roles for local staffs.Finally, although different solutions maywork for different synchrotron sources,any investment, given the current climatefor structural biology, would benefit alarge community of scientists.

Steven E. Ealick is in the Section ofBiochemistry, Molecular and Cell Biology,Cornell University, Ithaca, New York14853, USA. email: see3@cornell.edu

1. Smith, J.L. & Watenpaugh, K.D., eds., Structuralbiology and synchrotron radiation: assessment ofresources and needs, Structural BiologySynchrotron Users Organization (BioSync) (1991).

2. Smith, J.L., ed. Structural biology and synchrotronradiation: evaluation of resources and needs.Structural Biology Synchrotron UsersOrganization (BioSync), cited 17 June, 1998<http://www.ornl.gov/hgmis/biosync/> (1997).

important new projects quickly receivebeam time. Rapid access will require a fastefficient review process such as the web-based procedures that many beamlineshave already implemented. By combiningrapid review with regionally or nationallycoordinated scheduling it may be possibleto optimize the impact of structural biolo-gy. However, this may require the facilitiesto reconsider the emphasis placed onbuilding and maintaining a user base.

Young investigators. Another issue thatshould be addressed is how to assure thatyoung investigators, who have not yet hadthe opportunity to establish a record ofproductivity, gain access to synchrotronradiation. Even in cases where scientificmerit is clear, established investigatorswith equally meritorious projects mayhave an advantage because of existing pro-posals, track record and familiarity withthe system. It may be useful to consider apolicy of setting aside a fraction of beamtime for investigators who are first timeusers of a facility.

Remote access. Another mechanism forincreasing throughput at synchrotronbeamlines is to offer remote access.Because crystals can be frozen, pre-screened and shipped under liquid nitro-gen, it is possible for a user to carry outpreliminary examination of crystals or

By the late 1970s structural biology hademerged as a mature academic disci-pline. Dozens of macromolecular struc-tures had been determined by X-raydiffraction, and other techniques hadprovided additional structural know-ledge. An understanding of how thesestructures could be classified into cate-gories was beginning to emerge. Almostall of these efforts were being carried outin academic or governmental researchlaboratories. No significant investmentin structural research was being made in

industry because the case for its com-mercial utility had not been successfullyargued.

In the early 1980s pharmaceutical andchemical companies began to recognizethat structural research could, in fact,guide the development of certain prod-uct categories. One of these categorieswas industrial proteins, such as the bac-terial proteases used in laundry productsor xylose isomerase used in the produc-tion of high-fructose corn syrup, andcompanies interested in marketing pro-

teins for industrial applications recog-nized that they may need to alter thoseproteins to optimize their properties orto make them easier to produce in largequantities. The structures of native andaltered proteins could be determined tosee how optimized function or produc-tion had been achieved. These tech-niques, collectively known as proteinengineering, attracted a flurry of com-mercial interest around 1983. Only alimited degree of success has beenobtained from protein engineering

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efforts to date; perhaps the most visibleof these successes was thermal stabiliza-tion of subtilisin as used in laundrydetergents, accomplished at two biotech-nology companies in late 1980s.

The other products for which macro-molecular structures were seen to havecommercial relevance were pharmaceuti-cals. Most pharmaceutical compoundsare organic ligands that bind to macro-molecules in or on the surfaces of cells,and in general the macromolecules thatare targeted by pharmaceutical agents areassociated with diseases. Thus a drugmay have beneficial effects if it inhibitsthe action of an enzyme that is harmful,or if accelerates the action of a DNA-binding protein whose binding encour-ages expression of a useful gene. Usingmacromolecular structures as tools indesigning effective drugs can operate atseveral stages. If a pharmaceutical com-pany has no candidate drugs associatedwith a disease, then its scientists couldexamine the structure of the targetmacromolecule to choose an initial can-didate. If the macromolecule is anenzyme and its inhibition is the desiredendpoint, then they may examine theactive site to determine the shape andelectrostatic properties needed for aputative inhibitor. If instead a candidatedrug is already known, but its activity ortherapeutic index is too low, then theinteraction between the ‘lead candidate’and the macromolecule would be exam-ined to provide guidance on how toimprove on that lead candidate. Finally ifadequate activity has been achieved, per-haps through earlier iterations of design-ing inhibitors and determining thestructures of the inhibitor-enzyme com-plexes, further structure-based modifica-tions may be made to reduce the sideeffects of the drug or to increase itsbioavailability.

In the early 1980s several Americanand European pharmaceutical companiesresponded to this promise by hiringstructural biologists. In-house researchefforts in multi-dimensional nuclear reso-nance spectroscopy, molecular modeling,computational chemistry and macromole-cular crystallography were established atcompanies such as Merck ResearchLaboratories, the Upjohn Company, andAbbott Laboratories in the US; and atcompanies such as Hoffmann-La Roche,Wellcome Research, and Glaxo inEurope. A typical corporate commitmentinvolved hiring five scientists and buying$700,000 in crystallographic necessities

— X-ray generators, detectors, comput-ers, software, and protein purificationand crystallization supplies. Thecrystallographers worked on a wide variety of macromolecules, including nu-cleic acids, DNA-binding proteins,immunoglobulins and receptors, but thevast majority of the crystallographic pro-jects undertaken in industry were, andare, enzymatic, since the bulk of themacromolecules associated with diseasestates are enzymes. Generally a pharma-ceutical company’s scientists determinethe structure of the unliganded enzymeby conventional methods — multipleisomorphous replacement (MIR), mole-cular replacement and, increasingly,multi-wavelength anomalous diffraction(MAD). Once the native structure isknown, the corporate crystallographersproceed to determine the structures of awhole family of enzyme–substrate com-plexes, each illustrating the interactionof the enzyme with a drug candidate ormodel compound.

Industrial crystallographers atpeer-reviewed beamlinesThis initial commitment by industry toin-house crystallographic research coin-cided with the dramatic increase in theuse of storage-ring X-ray sources by aca-demic crystallographers. Unsurprisinglythere were tentative efforts made byindustrial crystallographers to takeadvantage of these regional or nationalresources, just as their colleagues incomputational chemistry took advan-tage of regional supercomputing centersfor their more ambitious ‘number-crunching’ efforts. An industrial macro-molecular crystallographer, having beenexposed to the advantages of storage-ring experimentation while in academia,would naturally want to exploit thoseadvantages in his or her new venue. Thehigh flux, fine collimation, and tunabili-ty of the storage rings are advantages forthe industrial scientists just as they arefor the academics.

However, for the industrial macro-molecular crystallographers, problemsquickly arose in the use of these regionalresources. Industrial scientists have tworequirements that do not arise in acade-mic research: the need for assuring con-fidentiality and the need for a rigorousmaintenance of intellectual propertyrights on the inventions that arise fromthe research. Without the former, theindustrial scientist risks being scoopedby the competition; without the latter,

the industrial scientist risks that patentscannot be enforced. Neither of theserequirements is easy to meet in an acade-mically operated regional research facili-ty like a storage ring. The kinds ofsecurity arrangements that maintain con-fidentiality within a corporate researchfacility are unavailable in a multi-institu-tion public facility. Ensuring intellectualproperty rights is difficult, because gov-ernments generally reserve some claim onintellectual property rights for researchresults obtained at publicly funded sites.These problems have muted the enthusi-asm of the structural biologists’ employ-ers for participation in these facilities.

Additionally, the technical needs of theindustrial scientist are not always identi-cal to those of the academic scientist. Incrystallography the differences are mat-ters of degree, but they can influence theways storage rings are used and the waysthat new beamlines are designed.Academic crystallographers generallydetermine unknown structures; indus-trial crystallographers spend the majori-ty of their time examining structures ofcomplexes between a protein of knownstructure and a small molecule ligand.These structures can be studied by dif-ference Fourier methods, and are there-fore simpler to execute and faster tocomplete than de novo structure deter-minations. Industrial crystallographersare expected to be productive; fifteenprotein–ligand structures per Ph.D. sci-entist per year is not unusual. The indus-trial crystallographer therefore needs tohave ‘well-oiled machinery’ for movingfrom availability of samples to finalrefined structures. A storage-ring beam-line with sophisticated tools for phasedetermination but poor user interfacesin its software will be less useful to theindustrial crystallographer than a slight-ly less advanced beamline where it is easyto move samples in and pull functionaldata out. These requirements are notincompatible with those of academiccrystallographers, but they are not iden-tical. If a beamline has been optimizedfor the requirements of academics, it willnot be precisely optimized for therequirements of industrial crystallogra-phers, and vice versa.

The final difficulty encountered byindustrial crystallographers in usingstorage-ring beamlines is access. Mostbeamlines dedicated to macromolecularcrystallography are over-subscribed by afactor of two, and the peer-reviewedapplication process does not provide

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reliable access to industrial users. Theindustrial crystallographers’ protein–lig-and structure studies are not as scientifi-cally dramatic as the de novo structuresthat are the mainstay of academiccrystallography and which sometimesinvolve developing new techniques thatare difficult to execute outside the storagering. Industrial scientists find it difficultto get substantial amounts of beam-timewhen the projects they propose underpeer review are less flashy than those oftheir academic counterparts.

The solution: custom beamlinesThe result of these difficulties is thatpharmaceutical crystallographers began,in the mid-1980s, to search for venues oftheir own to pursue their goals. Theysought administrative structures underwhich intellectual property rights couldbe guaranteed. They also sought the abili-ty to develop beamlines at least partlyunder their own control. These were to beoptimized to meet the technical needs ofindustrial crystallographers rather thanthose of academic scientists. With thesebeamlines, confidentiality could be insti-tutionalized and access by the corporatescientists would be guaranteed, indepen-dent of a peer review system. The admin-istrative groundwork was laid in theUnited States through discussions involv-ing officials of the Department of Energy(DOE), which operates all the American

second- and third-generation sourcesexcept CHESS, and industrial scientistsfrom Upjohn, duPont and other compa-nies. These discussions led to establish-ment of proprietary user agreementsbetween DOE facilities and individualcorporations under which the govern-ment would not, under defined circum-stances, interfere with the companies’intellectual property rights. The otherrequirements involved development ofindustrially-sponsored beamlines, andthose beamlines went into the planningstage in the late 1980s. The DOE stood tobenefit from these planned beamlines inthree ways. First, the industrial supportwould help to pay for facility develop-ment. Second, industrially-sponsoredresearch increases the breadth of repre-sentation in the facilities. Finally, theindustrial presence provides a public-relations boost to the DOE's efforts toappear as an active participant in eco-nomic development.

In that period Keith Watenpaugh ofUpjohn and Noel Jones of Eli Lilly & Co.recognized that a single corporation wasunlikely to make the financial commit-ment necessary for building a dedicatedindustrial beamline at a major facility(two to nine million dollars, dependingon the ambitiousness of the project andthe amount of infrastructure already inplace), whereas a consortium involvingseveral corporations could make such a

commitment. They discussed this concept with colleagues at other large pharmaceutical and chemical com-panies and later formed the Indust-rial Macromolecular CrystallographyAssociation (IMCA) to pursue the goal ofa pharmaceutical beamline at a storagering. They chose the Advanced PhotonSource (APS) at Argonne NationalLaboratory as the site for their facility,since the APS was then under construc-tion and promised the highest brillianceand the best opportunity for developingspecialized beamlines among Americansources. In 1992 IMCA contracted withthe Illinois Institute of Technology tobuild two beamlines for them: an inser-tion device beamline, optimized for monochromatic and multiwavelengthexperiments on small (<80 micron) pro-tein samples, and a bending magnetbeamline, optimized for monochromaticand multiwavelength experiments onlarger samples and for possible laterextension to polychromatic (Laue)experiments. The APS is structured intosectors, each consisting of an insertiondevice beamline and a bending-magnetbeamline, and there are substantial costsavings associated with developing anentire sector as a single administrativeunit; thus the decision to build out thepair of beamlines together was cost-effective. Design work for two beam-lines, one on a bending-magnet port and

Table 1 Involvement of pharmaceutical and chemical companies in crystallography1 at American storage rings

Company Location2 Storage Ring3 Beamline Organization3

Abbott Abbott Park, Illinois APS IMCABayer West Haven, Connecticut APS IMCABristol-Myers Squibb Princeton, New Jersey APS IMCAGlaxoWellcome Research Triangle Park, North Carolina APS IMCAEli Lilly Indianapolis, Indiana APS IMCAMerck & Co., Inc. Rahway, New Jersey and West Point, Pennsylvania APS IMCAMonsanto/Searle St. Louis, Missouri APS IMCAParke Davis Ann Arbor, Michigan APS IMCAPharmacia & Upjohn Kalamazoo, Michigan APS IMCAProcter & Gamble Cincinnati, Ohio APS IMCASchering-Plough Kenilworth, New Jersey APS IMCASmithKline Beecham King of Prussia, Pennsylvania APS IMCAHoffmann-La Roche Nutley, New Jersey NSLS X8CAmgen Thousand Oaks, California ALS MCFRoche Biosciences Palo Alto, California ALS MCFE.I. duPont de Nemours Wilmington, Delaware APS DND

1Listed are chemical and pharmaceutical companies that are contributing members of synchrotron beamlines at American storage rings. Not listedare companies whose scientists have collected diffraction data at academic or government-sponsored beamlines but whose corporations are notdirectly involved in beamline development or funding.2Location of organization from which the research originates; many of these companies have more than one site.3Abbreviations: APS: Advanced Photon Source, Argonne National Laboratory; NSLS: National Synchrotron Light Source, Brookhaven NationalLaboratory; ALS: Advanced Light Source, Lawrence Berkeley National Laboratory; IMCA: Industrial Macromolecular Crystallography Association;X8C: X-ray beamline 8C, National Synchrotron Light Source; MCF: Macromolecular Crystallography Facility, Advanced Light Source; DND: duPont-Northwestern-Dow Collaborative Access Team.

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one on an insertion-device port, beganimmediately, and construction began in1995. The first crystallographic demon-strations were made in late 1996 andpharmaceutically useful crystallographicdata began to emerge in mid-1997.IMCA now consists of twelve companies,listed in Table 1, and the IMCACollaborative Access Team (IMCA-CAT)comprises the IMCA companies and IIT.The IMCA beamlines are still under con-struction, but the companies are able touse more than 60% of the available beamtime on the IMCA-CAT insertion devicebeamline. The bending-magnet beam-line will become available for user exper-iments later this year. When both linesare available, each company will receivemore than three weeks of unrestrictedbeam time per year — substantiallymore than any company can obtain at apeer reviewed beamline. The IMCAcompanies can and do continue to col-lect synchrotron data at other facilitiesand at other APS beamlines.

Other pharmaceutical companies havesought involvement in other facilities.Many companies, notably the Europeanpharmaceutical companies and thebiotechnology companies on the westcoast of the US, have continued to useacademic beamlines at the EuropeanSynchrotron Radiation Facility, the APS,and several second-generation sources,competing for peer reviewed beam time.A group of Japanese scientists has nego-tiated to develop an IMCA-like arrange-ment at SPRing-8, and Hoffmann-LaRoche’s American crystallographershave become charter members of aPlanning Research Team (PRT) forbeamline X8C at the NationalSynchrotron Light Source (NSLS).Arrangements like these are likely tocontinue to develop, particularly as

research groups previously located atolder sources move their experiments tonewer facilities; as this occurs, beamlines‘open up’ at the older storage rings, andgroups partially funded from industrialsources can move in. Thus the opportu-nity to place an industrial stamp on theadministrative and technical fabric of abeamline can arise.

The futureIndustrial crystallography at storagerings will continue to grow, as drug com-panies expand their commitment tostructural biology and as appropriateadministrative and technical structuresare put in place at storage rings to facili-tate proprietary experiments. It will alsochange direction somewhat, as theunderlying science and the ways that itcan be applied change. The interest instructural genomics (see the report bySung-Hou Kim) extends to industry, andthere is likely to be an intense involve-ment by the pharmaceutical companiesin future plans for genomics-inspiredbeamlines. There are prospects for thedevelopment of new methods for deter-mining crystallographic phases, involving extensions of direct methodsto macromolecular crystallography.Industrial macromolecular crystallogra-phers will participate in the developmentand use of these experimental innova-tions, and if these changes have implica-tions to beamline design, the industrialscientists will play a role in specifyingthose designs.

An open question is where companieswill start new beamline projects. Someindustrial crystallographers prefer to col-lect synchrotron data close to their homebases, and that influences the way theyinvolve themselves in beamlines. Thusmost European companies have contin-

ued to collect data on peer-reviewedbeamlines at European synchrotrons, andbiotechnology companies on the westcoast of the US have generally resistedinvolvement in the APS and have concen-trated at the Stanford SynchrotronRadiation Laboratory (Stanford,California) and the Advanced LightSource (Berkeley, California). The IMCAcompanies are international in scope, butthe scientists who use the IMCA-CATbeamlines are concentrated in theAmerican midwest and northeast, lessthan three hours by air from the APS.There are notable exceptions — IMCAcorporate crystallographers from US sitescontinue to collect some data at Europeanstorage rings, and GlaxoWellcome scien-tists based in the UK and Pharmacia &Upjohn scientists based in Sweden havecollected data at the IMCA-CAT facilitiesat the APS — but for the most part indus-trial scientists are seeking regional solu-tions to their data collection needs. Thismay influence the way future beamlinesdevelop.

Regardless of where pharmaceuticalcompanies become involved in storage-ring science, they will most assuredly playsubstantial roles. Their commitment to theuse of structural results in drug design isdeep and continuing, and they recognizethat direct involvement in storage-ringfacilities will help them develop new drugs.

Andrew J. Howard is the Director ofIMCA-CAT at the Advanced PhotonSource, Argonne National Laboratory,Argonne, Illinois 60439 USA, and is in theCenter for Synchrotron RadiationResearch and Instrumentation, Biological,Chemical, and Physical SciencesDepartment, Illinois Institute ofTechnology, Chicago, IL 60616 USA.email: ahoward@psyche.csrri.iit.edu