Expression, purification, crystallization and preliminary X-ray diffraction analysis 2007

ActaCrystallographicaSectionF

StructuralBiologyandCrystallizationCommunications

ISSN1744-3091

Expression,purification,crystallizationandpreliminaryX-raydiffractionanalysisofThermotoganeapolitanab-glucosidaseB

󰀁-Glucosidasesbelongtofamilies1,3and9oftheglycosidehydrolasesandactoncello-oligosaccharides.Family1and3enzymesareretainingandarereportedtohavetransglycosylationactivity,whichcanbeusedtoproduceoligosacchar-idesandglycoconjugates.Family3enzymesarelesswellcharacterizedthantheirfamily1homologuesandtodateonlytwocrystalstructureshavebeensolved.Here,theexpression,purification,crystallizationandX-raydiffractiondataofafamily3󰀁-glucosidasefromthehyperthermophilicbacteriumThermotoganeapolitanaarereported.Crystalsofselenomethionine-substitutedproteinhavealsobeengrown.ThecrystalsbelongtospacegroupC2221,withunit-cell

˚.Nativedatahavebeencollectedtoparametersa=74.9,b=127.0,c=175.2A

˚resolutionandthestructurehasbeensolvedto2.7A˚usingthe2.4A

selenomethionineMADmethod.Modelbuildingandrefinementofthestructureareunderway.

PernillaTurner,aAnna

Pramhed,bErikKanders,aMartinHedstro¨m,aEvaNordberg

Karlssona*andDerekT.Loganb*

DepartmentofBiotechnology,CentreforChemistryandChemicalEngineering,LundUniversity,Box124,S-22100Lund,Sweden,andbDepartmentofMolecularBiophysics,

CentreforChemistryandChemicalEngineering,LundUniversity,Box124,S-22100Lund,Sweden

aCorrespondencee-mail:

eva.nordberg_karlsson@biotek.lu.se,derek.logan@mbfys.lu.se

Received2May2007Accepted14August2007

1.Introduction

Glycosidehydrolases(GH)areenzymesthathydrolyzeglycosidicbondsbetweentwoormorecarbohydratesorbetweenacarbo-hydrateandanoncarbohydratemoiety.Carbohydratesareessentialcomponentsofbiomass,whichisestimatedtobeproducedinaquantityofabout60GtyÀ1(Coxetal.,2000)andwhichcontainsanarrayofstructuralandstoragepolysaccharides.Toutilizetheserawmaterials,microorganismsproduceawidevarietyofcarbohydrate-hydrolyzingandcarbohydrate-modifyingglycosidehydrolases.Theseenzymescanalsobeusedasspecificcatalystsinindustrialapplica-tions,e.g.inthefoodandfeedindustries,thepaperandpulp,starchandtextileindustriesandinnewlyemerging‘green’processes(Turneretal.,2006,2007),takingadvantageoftheirspecificityinselectivepreparationsofcarbohydrate-containingrawmaterials.Basedonsequencesimilarities,GHhavetodatebeenclassifiedinto108separatefamilies(Coutinho&Henrissat,1999).󰀁-Glucosi-dases(EC3.2.1.21)playaroleinthecarbohydratemetabolismofmanyorganismsbyactingonthe󰀁-glycosidiclinkagesofcello-oligosaccharidescontaining󰀁-d-1,4-glycosidicbonds.TheseenzymesareclassifiedintothreeGHfamilies:GH1,GH3andGH9.BothGH1andGH3arefamilieswitharetainingmechanismandaredominatedbyenzymesactingonoligosaccharidesubstrates,whiletheGH9familyhasaninvertingmechanismandmostlycontainsendoglucanases.Retainingenzymesutilizeadouble-displacementmechanismwithretentionofconfigurationattheanomericcarbonofthesugarringandoftendisplaytransglycosylationabilities,whichcanbeofinterestforapplicationsfocusingonthesynthesisofoligo-saccharidesorrelatedproducts.Thecatalysisinvolvestwocarbox-ylateresidueslocatedonoppositesidesofthesugarplaneandthereactioncanbedividedintotwosteps:glycosylation,inwhichaglycosyl-enzymeintermediateisformed,anddeglycosylation,inwhichawatermolecule(hydrolysis)oranalcohol(transglycosyl-ation)hydrolyzestheglycosyl-enzyme(McCarter&Withers,1994;Sinnott,1990).Asfoldisbetterconservedthansequence,manyGH

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familieshavebeengroupedintostructurallyrelatedclans(GHA–N;Coutinho&Henrissat,1999).ClanAisbyfarthelargest,containing17GHfamiliesallsharingthe(󰀁/󰀂)8-fold,whiletheotherclans(GHB–N)onlycontaintwoorthreeGHfamilieseach.GH1󰀁-glucosi-dasesbelongtoclanAandhavebeenmorethoroughlycharacterizedthantheGH3representatives.Severalthree-dimensionalstructureshavebeensolved,someofwhicharefromthermophiles,e.g.a󰀁-glycosidasefromSulfolobussolfataricus(Aguilaretal.,1997)anda󰀁-glucosidasefromThermotogamaritima(Zecheletal.,2003).

EnzymesclassifiedasmembersofGH3donotbelongtoanyoftheknownGHclans,indicatingamoreunusualfold.Generally,knowl-edgeofthefunctionandstructureofGH3enzymesislessabundant,theirsequenceconservationisrelativelylowandfewenzymesarewellcharacterized.Forinstance,itispresentlystillimpossibletolocatetheacid/basecatalyticgroupsbasedonsequencehomology,aseventhisregionhaslowsequenceconservation.OnlytwocrystalstructureshavebeensolvedtodateofGH3enzymes:a󰀁-1,3-1,4-d-glucanexohydrolase(EC3.2.1.58)fromHordeumvulgare(barley;Vargheseetal.,1999;PDBcode1ex1)anda󰀁-hexosaminidase(EC3.2.1.52)fromVibriocholera(NewYorkStructuralGenomicsConsortium,unpublishedwork;PDBcodes1tr9and1y65).

TheenzymecrystallizedinthisworkoriginatesfromT.neapolitana(Tn)andisa󰀁-glucosidase(Bgl)classifiedintoGH3.TheenzymeisabbreviatedTnBgl3B,inaccordancewiththenomenclatureproposedbyHenrissatetal.(1998).ThegenewasisolatedfromDSMstrain4359withthepurposeofselectingacandidatecatalystforalkylglucosidesynthesisandtheenzymeshowedpromisingresultsinalkylglucosideproductionbytransglycosylation(Turneretal.,2007).ThestructurewiththehighesthomologytoTnBgl3B,thatofthebarleyenzyme,isonly20%identicaloverall;hence,structuralinformationonTnBgl3Bisessentialinordertogainfurtherknowl-edgeonthefunctionofthefamily3enzymes.Inaddition,TnBgl3BisthefirstthermostablerepresentativeofGH3,whichmayallowidentificationofthermostabilizingfeatures.

concentrationof1mMwhentheopticaldensityat620nmwas0.6andexpressionwasallowedtoproceedfor2h.

2.4.PurificationofSeMet-Bgl3B

Thecellculturewasharvestedbycentrifugationat5000gand277Kfor5min.Thepelletwasdissolvedin7mlbindingbuffer(20mMTris–HCl,0.75MNaCl,20mMimidazolepH7.5)andlysedbyultrasonicationinaUP400Sinstrument(DrHielscher,Stuttgart,Germany).Solubleproteinswereseparatedfromthecelldebrisbycentrifugationat80000gand277Kfor10min.Thesupernatantwaspassedthrougha0.45mmfilterandpurifiedona1mlHiTrapchelatingcolumn(GEHealthcare,Sweden).Thegelmatrixwaswashedwithdeionizedwaterbeforeloadingwithfivevolumesof5mgmlÀ1coppersulfate.Thecolumnwasthenwashedwithde-ionizedwaterandequilibratedwith10mlbindingbuffer.Crudeextractwasloadedontothecolumnandunboundproteinswerewashedoffwith10mlbindingbuffer.Elutionwasachievedwithagradientof20–500mMimidazolein20mMTris–HCl,0.75MNaClpH7.5.20fractionsof1mleachwerecollected.Thefractionscontainingproteinasobservedinthechromatogramoflightabsorptionat280nmwereanalyzedandusedforcrystallization.SDS–PAGEaccordingtoLaemmli(1970)wasusedtoanalyzetheenzymepurity.

2.5.Proteinanalyses

Inthestandardassayformeasuring󰀁-glucosidaseactivity,p-nitrophenolisreleasedfromp-nitrophenyl-󰀁-d-glucopyranoside(pNPG).40mlenzymesolutionwasaddedto96mlpreheated2.94mMpNPGdissolvedin20mMcitrate–phosphatebufferpH5.6andincubatedfor5mininaQBD2blockheater(Grant,UK)at358K.Afterincubation,thesampleswereputonicefor5minandtheabsorbanceat405nmwasreadusinganUltrospec1000spec-trophotometer(GEHealthcare,Sweden).Oneunitcorrespondstotheamountofenzymethatwillrelease1mmolp-nitrophenolperminuteunderthedescribedconditions.

2.6.Mass-spectrometricanalysisofSeMetincorporation

2.Materialsandmethods

2.1.Chemicals

AllchemicalswereproanalysifromMerckEurolabs(Darmstadt,Germany)unlessotherwisestated.

2.2.Expressionandpurificationofnativeprotein

His-taggedTnBgl3BwasproducedinEscherichiacolistrainTuner

´(DE3)growninminimalmediumbyfed-batchtechniques(deMare

etal.,2005).Extractsfromcultures,harvested4hafterinductionwithIPTG(isopropyl󰀁-d-1-thiogalactopyranoside),werepurifiedinatwo-stepprocedure.Heattreatment(343K,40min)precipitatedE.coliproteins,whichweresubsequentlyremovedbycentrifugation(27000g,30min).TheresultingsupernatantcontainingTnBgl3Bwasloadedontoanimmobilizedmetal-ionaffinitychromatographycolumnandpurifiedasdescribedpreviously(Turneretal.,2007).

2.3.SeMetincorporation

ThemolecularmassesoftheHis-taggedproteins(nativeandSeMet-modified)weredeterminedbyelectrosprayionizationmassspectrometryusingaQSTARhybridPulsariinstrument(AppliedBiosystems,CA,USA)equippedwithanano-ionsourcekit(Proxeon,Denmark).TheproteinsamplesweredesaltedusingaC4ZipTip(Millipore,MA,USA).5mlofthesolutionwasthenmixedwithanequalvolumeofacetonitrilecontaining0.1%formicacidbeforebeingappliedtothenanospraycapillary.Thenanospraysourcewassettopositive-ionmodewithasourcevoltageof+0.8kV.Thequadrupolesystemwasadjustedtoscanbetween800–3000m/zinTOF–MSmodeandchargeenvelopesoftheproteinvariantswereobtainedfrom120sofdataaccumulation.

2.7.Crystallization

Thebgl3b-containingplasmidwastransformedbyelectroporationintothemethionine-auxotrophicE.colistrainB834(DE3)(Novagen,Madison,WI,USA).Colonieswereinoculatedintominimalmediumwith25mgmlÀ1seleno-l-methionine(SeMet),cultivatedat310Kwithshakingforabout2dandthentransferredtoafresh200mlcultureofminimalmediumsupplementedwithSeMetandgrownat310K.ThecellswereinducedbytheadditionofIPTGtoafinal

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Purifiedproteinwasdialyzedagainst20mMMESbufferpH6.2andconcentratedto3–5mgmlÀ1.Suitableinitialproteinconcen-trationswerefoundusingthePre-CrystallizationScreen(PCT,HamptonResearch,CA,USA).InitialcrystallizationconditionswerefoundusingthePACTPremierscreen(MolecularDimensionsLtd,UK;Newmanetal.,2005).CrystallizationtrialsweresetupinGreinerlow-profile96-wellplatesusingaMosquitorobot(TTPLabtech,UK)at293K.Dropsconsistingof100nlproteinsolutionmixedwith100nlreservoirsolutionwereequilibratedagainst80mlreservoir

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solution.Paralleltrialsweresetupforthenativeproteinataconcentrationof5mgmlÀ1andfortheSeMetproteinat3.1mgmlÀ1.Smallcrystalsofbothproteinsappearedafter4dindropswheretheprecipitantwas20%PEG3350in0.1Mbis-TrispropanepH7.5andanyof0.2MNaBr,NaI,KSCNandNa2SO4.ThemostpromisingdropswereoptimizedwithrespecttoPEGandsaltconcentration.Thecrystalsusedfordatacollectiongrewinhangingdropsconsistingof1mlproteinsolutionand1mlreservoirsolutionequilibratedagainst1mlreservoirsolution.Thebestcrystallizationconditionsfor

Table1

X-raydata-collectionandphasingstatistics.

Valuesinparenthesesareforthehighestresolutionshell.

SeMetPeak

Datacollection

Unit-cellparameters

˚)Wavelength(A˚)Resolution(A

Rmerge†(%)

TotalobservationsUniquereflectionsAverageredundancy‡Completeness(%)hI/󰀃(I)iSnorm/Sano§

˚)Phasing(to2.7A

Phasingpower(dispersive/anomalous)RCullis}(dispersive/anomalous)

Meanfigureofmerit(centric/acentric)

a=74.9,b=127.0,c=175.20.97909

24–2.7(2.87–2.7)6.3(43.7)102212433102.4

97.6(97.4)10.7(2.1)1.16(1.04)—/0.813—/0.862

Inflectionpoint

a=74.9,b=127.0,c=175.20.97924

24–2.7(2.87–2.7)6.4(41.1)99797426442.3

97.2(96.0)11.0(2.1)1.15(1.03)0.071/0.8060.678/0.8680.168/0.358

High-energyremotea=74.9,b=127.0,c=175.20.97565

19–2.9(3.07–2.9)7.9(46.3)82094362272.3

97.3(97.5)9.9(1.8)1.07(1.04)0.218/0.5880.876/0.963

Native

a=74.9,b=127.2,c=175.20.97906

40–2.4(2.5–2.4)6.4(45.3)143480327994.4

98.9(98.2)16.7(3.7)1.02(1.02)

thenativeproteinwere16–20%PEG3350,0.1–0.2MNaIandfortheSeMetproteintheywere20–24%PEG3350,0.1–0.25MNaI,bothin90mMbis-TrispropanebufferpH7.4.

2.8.Datacollection

Allcrystalswerecryoprotectedwith25%glycerol,20%PEG3350,0.2MNaI,90mMbis-TrispropanepH7.4andflash-cooleddirectlyinaliquid-nitrogenstreamfromanCryostreamcooler(OxfordCryo-

PPPP

†Rmerge(I)=jkjIjkÀhIijj=jkIjk,whereIjkarethekindividualobservationsofeachreflectionjandhIijisthevalueafterweightedaveraging.‡FriedelmatesPParetreatedasseparatereflectionsfortheMADdataset.§S/S=h󰀃(I)iassumingFriedel’slawtobetrue/h󰀃(I)iassumingFriedel’slawtobefalse.}R=\"=ÁisoforacentricnormanocullisisoPP

reflectionsand\"ano=ÁBijvoetforanomalousdifferences,where\"isoand\"anoaretheisomorphousandanomalouslackofclosure,respectively,ÁisoistheisomorphousdifferenceandÁBijvoetistheBijvoetdifference.

Figure1

Amino-acidsequencealignmentofglycosidehydrolasefamily3.Theregionssurroundingtheputativeactive-siteregionareshown.Abbreviations:TnBglB,ThermotoganeapolitanaDSM4359󰀁-glucosidase3B;TmBgl,T.maritimaMSB8󰀁-glucosidase;FnGH,Fervidobacteriumnodosumglycosidehydrolase;AhBgl,Aeromonashydrophila󰀁-glucosidase;FspBgl,Flavobacteriumsp.MED217󰀁-glucosidase;PaBgl,Prevotellaalbensis󰀁-glucosidase;YpBgl,YersiniapestisNepal516󰀁-glucosidase;CtBglB,Clostridiumthermocellum󰀁-glucosidaseB;CsBgl,C.stercorarium󰀁-glucosidase;PspGlc,Paenibacillussp.TS12glucocerebrosidase;AnBgl,Aspergillusniger󰀁-glucosidase;FmBgl,Flavobacteriummeningosepticum󰀁-glucosidase;HvBgl,barley󰀁-d-glucanexohydrolaseExoI.Aminoacidsinboldhavebeendeterminedexperimentallyascatalyticaminoacids,aninvertedtriangledenotesthepositionofthenucleophileanddiamondsdenotethepositionsofverifiedacid/baseresidues.

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systems,UK).Thecrystalscouldbesoakedinthecryosolutionforaperiodofafewsecondstoafewminuteswithoutanyobservabledifferenceindiffractionquality.Crystalswerepre-screenedatstationI911-5oftheMAX-IIsynchrotron(Lund,Sweden).Three-wave-˚resolutionwerecollectedatstationlengthSeMetMADdatato2.7A

ID29attheESRFsynchrotron(Grenoble,France)fromacrystalmeasuringapproximately400Â80Â20mmgrownin20%PEG3350,0.15MNaI,90mMbis-TrispropanepH7.4.Theoscillationrangewas0.7󰀄and160imageswerecollectedforeachwavelength.Anative

˚resolutionwascollectedfromacrystalmeasuringdatasetto2.4A

approximately400Â200Â20mmgrownin16%PEG3350,0.1MKBr,90mMbis-TrispropanepH7.4.Theoscillationrangewas0.5󰀄and244imageswerecollected.Thebeamsizewas50Â50mmandtherod-shapedcrystalsweretranslatedtoexposeafreshvolumeaftereachwavelength(fortheMADdata)andonceduringeachwavelength.SeveralMADdatasetswerecollectedfromseparatecrystalsandthatproducingthebestoverallfigureofmeritfortheexperimentalphaseswasusedforstructuredetermination(Table1).DatawereintegratedandscaledusingXDS(Kabsch,1993,2001).FurtherdatareductionandmanipulationusedtheCCP4package(CollaborativeComputationalProject,Number4,1994)driventhroughtheCCP4iinterface(Pottertonetal.,2003).TheanomalousscatteringsubstructurewassolvedusingautoSHARP(deLaFortelle&Bricogne,1997),exploitingSHELXDforPattersonfunction

´netal.,2003).Phaseswereimprovedbysolventflippingsolution(Uso

usingSOLOMON(Abrahams&Leslie,1996).Theoptimalsolventcontentforthisprocesswas47.8%.

ofTnBgl3Bis96%identicaltoa󰀁-glucosidaseisolatedfromadifferentstrainofthesamespeciesandthemajordifferenceisanonconservedstretchclosetotheN-terminus(Zverlovetal.,1997).TheTnBgl3BsequencewasalignedagainstanumberofothersequencesfromtheGH3family(Fig.1).Theoverallsequenceidentitywiththestructurallydeterminedbarleyenzyme(HvBgl)isonly20%.Distinctphylogeneticclustersofenzymeswithinthefamilyhavebeenidentified,withsixmajorbranches(Harveyetal.,2000).TnBgl3Bislocatedincluster5,togetherwithenzymesfromT.maritima(AE001690),Prevotellaruminicola(U35425),Clos-tridiumthermocellum(X15644),C.stercorarium(Z94045)andRuminicoccusalbus(U92808).TnBgl3BisrelativelydistantfromtheH.vulgareenzyme,whichisfoundincluster1togetherwithrepre-sentativesfromanumberofotherplants.Alignmentsalsoshowedadifferenceinthelengthofthesequencebetweenthesetwoenzymes,withthethermostableTnBgl3BhavingaC-terminalextensionofapproximately115aminoacids,indicatingadifferenceinthenumberofdomains.Thisfindingisalsosupportedbyclusteranalysis(Harveyetal.,2000).

3.2.ProductionandSeMetincorporation

3.Resultsanddiscussion

3.1.Overallsequence

Glycosidehydrolasefamily3includes873genesequences,ofwhichalmostallarebacterial(631)oreukaryotic(236).Despitethelargenumberofsequences,ratherfewGH3enzymeshavebeenbiochemicallycharacterized.Onlyone,thatfromH.vulgare,hasbeencharacterizedatthestructurallevel(Vargheseetal.,1999;Hrmovaetal.,2004,2005).

GHfamily3alsoincludesthermostableenzymesandinthisworkwehavecrystallizeda󰀁-glucosidaseoriginatingfromthehyperther-mophileT.neapolitana(DSMstrain4359).Theamino-acidsequence

ProductionoftheHis-tagged󰀁-glucosidaseinE.coliTuner(DE3)[maximumspecificgrowthrate(󰀄max)=0.7hÀ1at310K]resultedinanintracellularrecombinantproteinlevelcorrespondingtoapproximately15%ofthetotalprotein.

Themethionine-auxotrophicstraingrewslowly(󰀄max=0.3hÀ1at310K),buttheSeMet-substitutedTnBgl3Balsoconstitutedapproximately15%ofthetotalprotein.BoththeSeMet-substitutedandthenativeglucosidaseweresuccessfullypurifiedtoabout90%(Fig.2)andtheactivityofthepurifiedsampleswasverified(datanotshown).

SeMetincorporationwasconfirmedbynanospraymassspectro-metry.PurifiedsamplesofHis-taggednativeandSeMet-TnBgl3Bwereexaminedandtheaveragemasseswere82225and82740Da,respectively.Theadditionalmassof515Dacorrespondstothedifferenceobtainedwhenseleniumreplacessulfurin11methionineresidues,thetotalnumberinthesequence.SeMetthereforeoccupiesallpossiblemethioninesitesintheprotein.

3.3.Crystallizationanddatacollection

BothnativeTnBgl3BandSeMet-TnBgl3BcrystallizedinthePACTPremierScreenandtheconditionswerefurtheroptimized.CrystalsbelongtospacegroupC2221,withtheunit-cellparameters

Figure2

SDS–PAGEanalysisofproteinpurifiedbyIMAC.(a)SeMet-TnBgl3B,(b)TnBgl3B.

Figure3

AnativeTnBgl3BcrystalintheX-raybeamatbeamlineID29oftheESRF.Thelengthofthecrystalisapproximately400mm.

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WethankthebeamlinestaffatMAX-labandtheESRFforassistancewithdatacollection.TheB834(DE3)strainwasakindgiftfromDrClaesvonWachenfeldt,DepartmentofCellandOrganismBiology,LundUniversity.FinancialsupportfromTheFoundationforStrategicEnvironmentalResearch,MistratoENKandPTandfrom

˚det)toENKandDListheSwedishResearchCouncil(Vetenskapsra

gratefullyacknowledged.ENKalsoacknowledgestheKrapperupFoundationforadditionalsupport.

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Figure4

FirstdiffractionimagefromthenativeTnBgl3Bcrystal.Thebluecircleindicatesa

˚.resolutionof2.3A

giveninTable1.ThereisoneTnBgl3Bmoleculeintheasymmetric

unit,givingasolventcontentof57%.

Thecrystalsusedfordatacollectionwereplate-shapedandhaddimensionsofaround300Â200Â20mm(Fig.3).X-raydatawere

˚fortheSeMetproteinand2.4A˚forcollectedtoaresolutionof2.7A

thenativeprotein(Fig.4andTable1).

PhasingstatisticsarepresentedinTable1.Theanomaloussignal

˚wasweak:autoSHARPsuggestedresolutioncutoffsof3.67and4.1A

forthepeakandinflection-pointdata,respectively,despitea

˚.TenSepositionswerefoundanddiffractionresolutionlimitof2.7A

refinedtooccupanciesof0.4–0.9.Thelowoccupancieswereun-expected,astheincorporationofSeMethadbeenshowntobe100%bymassspectrometry.However,twodifferentbatchesofproteinwereused:oneforcheckingtheincorporationofSeMetandoneforgrowingthecrystalsfromwhichtheX-raydatawerecollected.ThesameprotocolforSeMetincorporationwasused,butthereprodu-cibilityofincorporationwasnottested.Someofthemethionineresiduesmaybeonthesurfaceandthuslesswellordered.Never-theless,theelectron-densitymapsaftersolventflatteningwereofgoodqualityandmanualmodelbuildingandrefinementareinprogress.

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