Laboratory exercises for protein crystallization
Transcript of Laboratory exercises for protein crystallization
Laboratory exercises for protein crystallization – 3 HEC PhD course PNS0158 Protein crystallization
and X-ray data collection 9-20 April 2018
9-13 April 2018
Terese Bergfors, Uppsala University
Jerry Ståhlberg, SLU
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Tableofcontents
TuesdayExo1.Lysozyme:hangingandsittingdropsExp.2.CBH1:gridscreenandcrystallizationWednesdaya.m.Exp.3.DemoofmatrixmicroseedingatBMCWednesdayp.m.Exp.4.CBH1:manualmicroseedingExp.5.ConstructingaphasediagramwithlysozymeExp.6.Saltorproteincrystal?TwosimpletestsThursdayExp.7.Lysozyme:ligandsoakingwitheosinredExp.8.CBH1:ligandsoakingbythreemethodsExp.9.PracticesessionwithcrystalharvestingandcryocoolingFridaya.m.Exp.10.CBH1:cryocoolinganddewarstorage
Theroadofinstructionislongbymeansoftheoriesbutshortandefficientthrough
examples.
Seneca,5B.C.–65A.D.
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TuesdayExercise1.Vapordiffusionwithhangingandsittingdrops:manualsetupwithlysozyme. Materials• One24-wellplate• 6coverslipsforthehangingdrops• clearsealingtapeforsittingdropexperiments• 6sitting-dropbridges• greaseforthecoverslips(forthehangingdrops)• pipettesProteins• Lysozyme20mg/mlPrecipitantSolutions• 5,10,15,20,25,30%NaClin0.1Msodiumacetatebuffer,pH4.7Hereisthebasicprocedureforamanualsetupin24-wellplates.Hangingdrops
1. Choosea24-wellplatewiththickrimsaroundthereservoirsandgreasetherims.(Itisalsopossibletobuypre-greasedplates).
2. Pipete0.5mloftheprecipitantsolutionintothereservoiroftheplate.3. Placeasilanizedcoversliponthebenchtop.Carefullypipet1-5
microlitersofproteinsolutionontothecoverslip.4. Add1-5microlitersofthereservoirsolutiontotheprotein.Avoidbubbles
andmakethedropassphericalaspossible.5. Optional:mix/stirthedrop.Mixingincreasesthenucleationrate.This
mayormaynotbeaneffectyouwant.Thechoiceisyours,butbeconsistent.Don’tmixsomedropsandnottheothers,orviceversa.
6. Withapairoftweezers,invertthecoverslipandsuspenditoverthereservoirsolutionbyplacingthecoverslipontothegreasedrim.Pressgentlytoensureagoodseal.
7. Preparetherestoftheplateinthesamemanner.
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Sittingdrops1. Sittingdropplatesarecommerciallyavailableoryoucaninsertsitting-
dropbridgesintoanordinary24-wellplate.2. Putthescreeningsolutionintothereservoir.3. Pipettheproteinintothesitting-dropdepression.4. Addthereservoirsolutionasabove.5. SealtheexperimentwithClear-Sealtape.
Today'sexperimentTheproteinconcentrationwillbekeptconstant(20mg/ml)andwewillvarytheprecipitantconcentrationfrom5to30%NaClin50mMsodiumacetate,pH4.7.Makedropsof2microlitersproteinplus2microlitersreservoirsolution.Onerowwillbehangingdrops.Theotherrowwillbethesame,butsittingdrops.Thelayoutwilllooklikethis:
5% 10% 15% 20% 25% 30%hangingdrops
sittingdrops
Questionsfordiscussion
1. Inaninitialscreeningexperiment,whichmakesmoresensetodo:setupthesamekitashangingandsittingdropsorsetuptwodifferentkitsassittingdropsonly?
2. Whyandwhendopeopleusehangingdrops?Whichdoyouprefer?3. Whichkindofdropisbettertosetupat4degrees?
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TuesdayExercise2.OptimizationofcrystallizationconditionsforCBH1bygridscreening,andcocrystallizationwithligand,usingthehangingdropmethodin15-wellplates. Materialsandsolutions• One15-wellplatewithscrewcaps(EasyXtal;Qiagen)• stepperpipettewithpositivedisplacementtips• pipettes,downto1ulvolume• 50%mPEG5000(polyethyleneglycolmonomethylether)=precipitant• 1MNa-MES(sodium2-(N-morpholino)-ethanesulfonicacid),pH6.0=buffer• glycerol(>95%)=cryoprotectant• 1Mcobaltchloride,CoCl2=metaladditive• CBH1protein,~10mg/ml,in10mMsodiumacetate(NaAc)bufferpH5.0• ligandsolution,5mMcellobiose(preliminary)ProcedureEachgroupshallsetup9crystallizationdropsinasmallgridscreen: 1 2 3 4 5 2mg/ml
protein8mg/mlprotein
8mg/ml+ligand
empty empty
A:15%mPEG300ul50%515ulwater
B:17.5%mPEG350ul50%465ulwater
C:20%mPEG400ul50%415ulwater
1mlreservoirsinthewells:15%,17.5%or20%mPEG5000+50ul1MNaMespH6.0→50mM+125ulglycerol→12.5%+10ul1MCoCl2→10mMCrystallizationdrops:1+1ul
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Mixreservoirs1. Takeone15-wellplate.Markwithdate,experiment,groupnumberand
names.Youmayalsoscribbledirectlyontheplatewhatyouhaveineachrow/columntoquicklyseewhatiswhere.
2. Pipetwatertothewells,515ulinrowA,465ulinrowB,415ulinrowC.3. Add10ulCoCl2and50ulNaMESbuffertoeachwell.4. Useastepperpipettetoaddglycerol(125ul),andmPEG(seevolumes
above).NB!Ordinarypipettesdonotworkwellwiththeseviscoussolutions.
5. DonotworryaboutprecipitationofmPEGinthewells.Shaketheplatesomeminutesontheplateshakeruntilreservoirsareclear.
Hangingdrops
6. Pipet8ulwatertoaneppendorftube.Add2ulCBH1stocksolution,todiluteto2mg/ml,touseforcolumn1.
7. Pipet2ulwatertoaneppendorftube.Add8ulCBH1stocksolution,todiluteto8mg/ml,touseforcolumn2.
8. Pipet2ulligandsolutiontoaneppendorftube.Add8ulCBH1stocksolution,todiluteto8mg/ml,touseforcolumn3.
9. Organizeallyouneedforsteps10-12,sothatyoucansetupthecrystallizationndropsswiftly,inordertopreventsubstantialevaporationfromthedrops.
10. Place1ulofthecorrectproteinsolutiononascrewcap.Avoidairbubbles.
11. Keepthepipettetip,andtake1ulfromthecorrespondingreservoir/wellandaddtotheproteindrop(nomixingisneeded).Avoidairbubbles.
12. Turnthescrewcapupsidedownsothedropwillbehanging.Placeabovethecorrespondingwell.Screwtofastenandtightengently.
13. Preparetherestofthedropsinthesamemanner.14. Examineyourdropsunderthemicroscopeandnoteifyouhavealready
gotsomecrystals,orprecipitate,orifthedropsareclear.Youmay,e.g.,usethetableaboveforyournotes.
15. Puttheplateattheplacededicatedforcrystallizationexperimentsuntiltomorrow.
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WednesdaySeeding
SeedingLabExercisesBackground
1.Whatisseeding?
• Theuseofanexistingnucleus(usuallyasmallcrystalorcrystalfragment),
introducedintoanewdrop,whereitactsasagrowthsite.
2.Whyseed?
• Toseparatenucleationfromgrowthandbypasstheneedforspontaneous
nucleation.Itiseasiertoaddontoanalreadyexistingnucleusthancreate
onedenovo.SeeFig.1.Seedingandthephasediagram.
• Improvethesizeofthecrystalsorcontrolthenumberofcrystals
• Getmoreconsistentresultswhencrystalsdon’talwaysappearinknown
conditions
• Speedupresultsifspontaneousnucleationisslow
• Toobtainawiderrangeofcrystalforms(polymorphs)byseedingintototally
differentprecipitants
Ifyoucanlookintotheseedsoftimeandsaywhichgrainwillgrowandwhichwillnot,speak
thentome....
MacbethAct1,Scene3
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3.Whatarethetypesofseeding?
• Macroseedingisthetransferofasingle,pre-grown,washedcrystal.
• Microseedingisthetransferofmicroscopiccrystals,crushedupinto
fragments.
• Streakseedingisaformofmicroseedingthattransfersthemicroseedsbya
strokingmotionwithawhiskerorhairofsomesort.Canbeusedonitsown
orincombinationwithmicroseedsinadilutionseries.
• Jabseedingisanothervariationofmicroseedingwherethenewdropis
“innoculated”withasinglejaboftheseedtransfertool.
• Matrixmicroseedingiswhenmicroseedsareplacedintoascreenof
conditionstotallyunrelatedtotheoneswheretheseedcrystalsoriginated.
Reference:
Forareviewarticleonseeding,see:Bergfors,T.“SeedstoCrystals”J.Structural
Biol.2003,vol.142,66-76
Foranarticleonmicroseedmatrixscreeningsee:D'Arcy,Bergfors,Cowan-Jacob
&Marsh."Microseedmatrixscreeningforoptimizationinproteincrystallization:
whathavewelearned?"ActaCryst2014,F70,1117-1126.Thisisthemethodwe
willdemonstrateforyouattheBMCB7:205labonWednesday.
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Wednesdaya.m.Exp.3.DemoofmatrixmicroseedingVisittoBMCB7:205,Husargatan3https://www.google.se/maps/@59.8418476,17.6348823,17.99zWednesdayp.m.Exp.4.CBH1:manualmicroseedingMaterialsandsolutions• Pipettes,downto1ulvolume• solutionforseedpreparation=precipitantsolutionwith20%mPEG5K,50mM
Na-MESbufferpH6.0,10mMcobaltchloride,and12.5%glycerol(provided)• onecrystallizationdropwithCBH1crystals(NB!Notcrystalsofactivewildtype
CBH1ifyouarecrystallisinganinactivemutant)• plate(s)withcrystallizationdropstobeseeded,i.e.theCBH1platethatyouset
upyesterday(onTuesday)• acupunctureneedles(forseeding)• pipettesMicroseedsuspension
1. Examinethecrystallizationplatefromyesterdayunderthemicroscope.Noteifyouhavealreadygotsomecrystals,orprecipitate,orifthedropsareclear.
2. Findandselectonecrystallizationdropwithcrystalstouseforpreparingmicroseeds,amongyourownifyoualreadygotcrystals,orpregrowncrystalsprovidedbytheteacher.Donottakeadropwhereyouhavecrystalsthatmaypotentiallybeusedfordatacollection,butonewheretheyarelessnice-looking.
3. Pipet~200ulofthesolutionforseedpreparationtoaneppendorftube.(Alternativelyyoucantake~200ulofreservoirsolutionofthechosendrop).
4. Pipetsome5-10ulfromtheeppendorftubetothedropwithcrystals.Suckitallupagain,withcrystals,andaddtothesolutionintheeppendorftube.
5. Vortexthetube30-60seconds.6. Centrifuge1-2minutesatmaxspeed(e.g.13,000rpm)inaneppendorf
centrifuge.7. Transfersome100-150ulofthesupernatanttoaneweppendorftube,
beingcarefulnottostirupanypelletedstufffromthebottomofthetube.Thesupernatantisyourmicroseedsolution(orrathersuspension),readyforuse.
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Microseedingwithneedle8. Pipet100-500ulofsolutionforseedpreparationasreservoirinanempty
wellonaplate.9. Pipetsome5-10ulofyourmicroseedsolutiononanewscrewcap.Place
ontopofthewellwiththereservoir.Donottightenthecapatthisstage.10. Screwoffthecapwiththecrystallizationdroptobeseeded,turnupside
down,andplaceinfrontofyou(orholdwithonehandwhileseedingwiththeotherhand).
11. Dipanacupunctureneedleinthemicroseedsolutiondrop,thenjusttouchthesurfaceofthecrystallizationdropwiththetipoftheneedle.Thelessoftheneedlethatisimmersed,thebetter.
12. Putbackthescrewcapaboveitswell.13. Repeattheprocedurewithallthecrystallizationdrops.Youcanusethe
sameneedleforalldrops.Shouldyourinsetheneedleoffinbetweenjabs?It'suptoyou,butamakeanoteofwhetherornotyoudoit.Itmayincreasethenumberofseedsinthesubsequentdropsifyoudon'trinsebetweenjabs.Thismayormaynotbeagoodthing.Itdependsifyouhavetoomanyortoofewseedstobeginwith.So,asalwaysinlabwork,keepcarefulnotesofwhatyouhavedone.
14. Takeaquicklookattheseededdropsinyourplate.Noteany“new”observations(e.g.precipitate,dust,dirt,airbubblesetc).
15. Puttheplateattheplaceforcrystallizationexperimentsuntiltomorrow.
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WednesdayExp5.Phasediagramwithlysozyme:Determinationofthesupersolubilitycurveinamicrobatchsetup.
Purposeofthisexperiment:Inthisexperimentwewillconstructatwo-dimensionalgridscreenwherewevarytheproteinconcentrationvs.thesaltconcentration.Youwanttofindthenucleationzone,becausethisiswherecrystalsfirstnucleate.Tofindthisregion,wewillempiricallyconstructatwo-dimensionalphasediagram(proteinconcentrationvs.precipitantconcentration)forlysozyme.Within30minutesorless,wecanestablishtheprecipitationboundary.Ifyoucontinuetheexperiment24-48hours,itisalsopossibletomapthenucleationzone.Knowingbothoftheseallowsustoplotthesupersolubilitycurve.Itisassumedyouknowthedifferentregionsofthephasediagram.Ifnot,heretheyareagain(seeFig.1).
Figure1.Aschematicshowingthedifferentregionsofthephasediagram.Theexactlocationsofthesezonesmustbedeterminedempiricallyforeachproteinandprecipitant.Reprintedhereforteachingpurposeswithpermissionfromtheauthor,N.E.Chayen,reference1.
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Materialsrequired:• Terasakiplate• Paraffinoil(about8mlisrequiredtofilltheTerasakiplate).• P2Gilsonpipetteorsimilar
Solutionsandproteinsrequired:• 5%,10%,15%,20%,25%,and30%NaClbufferedin0.1MNaacetate,pH4.7.
Usethesolutionsfromyesterday,Exp.1.• lysozymewitheosinred,at10,20,40,60,80,and100mg/ml,dissolvedin
water.Thesedilutionshavealreadybeenpreparedforyou.Procedure:
1. FloodtheTerasakiplatewiththeparaffinoil,i.e.,about8ml.Makesureeachwellisflooded.
2. Pipette2microlitersofeachNaClsolutionacrossasshowninthediagrambelow,Fig2.
3. Pipette2microlitersofeachlysozymeconcentrationasshowninFig.2.4. Stirthedropssothattheymix.5. Optional(ifyouhaveenoughtime):Repeatthegrid(thereshouldbeenough
roomonthesameTerasakiplatebyusingrows7-12),butthistimestirallthedrops.Alternatively,yousetuponeexperimentwhichyoudonotstirbutyourlabpartnersetsupthesameexperimentandstirshis/herdrops.
6. Wait5-30minutes.Plottheresultsasatwo-dimensionalgraph:proteinvs.precipitantconcentration.
7. Optional:Wait24-48hours.Plottheresultsagain.(Intoday’sexperimentwewillonlydosteps1-6.)
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Fig.2.TerasakiplatesetupThedropletsinrows1-6shouldbestirred.Avoidintroducingairbubblesifyouaspiratethesolutionsupanddown.
5%NaCl
10%NaCl
15%NaCl
20%NaCl
25%NaCl
30%NaCl
Row1 100mg/ml
Row2
80
Row3
60
Row4
40
Row5
20
Row6
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A B C D E F
Expectedresults:
• Youshouldseetwoclearlydelineatedzoneswheresomedropsstayclearandotherdropsprecipitatemoreorlessimmediately(<30minutes).Thisgivesyoutheprecipitationzone.
• After24-48hoursyoushouldclearlyseewhichconditionsleadtonucleationwithsubsequentcrystalgrowthandwhichonesremainclear.Thezonewithcrystalsisthenucleationzone.Thecleardropsareeitherundersaturatedorinthemetastablezone.
• Ifyouplotthenucleationandprecipitationzones,youcanestablishthesupersolubilitycurve.
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• Todistinguishtheundersaturatedzonefromthemetastableone,onecouldstreakseedallthecleardrops.Dropsthatcansupportcrystalgrowth,butdonotgiverisetoitspontaneouslyaremetastable.Ontheotherhand,iftheseedsdissolve,thedropisundersaturated.Ifyouweretocollectallthisinformationyoucouldnowconstructacompletephasediagram,showingallthedifferentzonesinFigure1.
Discussionpoints:Whydeterminetheprecipitationboundaryandsupersolubilitylimit?
1. Crystallizationusuallyoccursclosetothesetwozones.Ifyoucompareyourfirstplot(theonemadeafter30minutes)andagainafter48hours,youwillclearlyseethatnucleationleadingtocrystalformationdoesinfactoccurclosetotheprecipitationboundary.
1. Asupersolubilitylinecanbedeterminedwithrelativelylittlematerialand
quitequickly.ThisiswhatSaradakisandChayencalla“workingphasediagram”(ref.2).Itisnotacompletephasediagram,butithasenoughinformationsuchthatyoucandesignusefulexperimentsfromit.Youcanusetheinformationobtainedforfurtherscreeningsetupsoroptimization.Formoredetailsonputtingthisexperimentintopractice,seereferences3-5.
2. ForagoodexplanationofWHYyouwanttodothis,insteadofcarryingon
withsparsematrixscreening,seereference6.ToquoteHansenetal.(seereferencebelow),“…achievingoptimallevelsofsupersaturationismoreeffectivethanbroadsamplingofchemicalspace.”Ifyouarenotgettingresultswithyourusualsparsematrixtypescreening,considerthemethodpresentedinthislabexerciseasanalternative.
References
• Chayen,NE.2005.Methodsforseparatingnucleationandgrowthinproteincrystallization.ProgBiophysMolBiol88,329-337.
• Saridakis,E.,Chayen,NE.2003.Systematicimprovementofproteincrystalsbydeterminingthesupersolubilitycurvesofphasediagrams.BiophysJ84,1218-1222.
• ShawStewart,PD,Khimasia,M.1994.Predispensedgradientmatrices-anewrapidmethodoffindingcrystallizationconditions.ActaCrystD50,441-442.
• Douglasinstrumentsapplicationnotes.Crystallizationofaproteinbymicroseedingafterestablishingitsphasediagram.http://www.douglas.co.uk/rep1.htm
• Hansen,CL,Sommer,MO,Quake,SR.2004.Systematicinvestigationofproteinphasebehaviorwithamicrofluidicformulator.PNAS101,14431-14436.
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WednesdayExercise6.Saltorproteincrystal?TwosimpletestsBackground:reprintedfrom:Bergfors,T.inMethodsinMolecularBiology,vol.363:MacromolecularCrystallographyProtocols:Volume1:PreparationandCrystallizationofMacromoleculesEditedby:S.Doublié©HumanaPressInc.,Totowa,NJ.
Manyofthecomponentscommonlyusedforcrystallizingproteincanalsogiverisetosaltcrystals.Thereforenocrystalshouldbecelebrateduntilitisknownifitissaltorprotein.Methodsfortestingifacrystalissaltorproteininclude:1.X-raydiffractionThisisthedefinitivemethodfordeterminingifacrystalissaltordiffractionprotein.Ifthecrystalcanbemounted,checkthediffractionpatternonanin-houseX-raysource.Theanswerwillbeimmediatelyobviousbecausethediffractionspotsforsalt(athighresolu-tion)arefewandfarapart,whereastheyaremanyandclosetogetherformacromolecularcrystals.Collecta3–5°oscillationpicturewiththedetectorclosetothecrystal.
Leftimage:proteindiffraction.Rightimage:saltcrystalwithicerings.(leftimagecourtesyofC.Björkelid,UppsalaUniversity)2.SnaptestWiththedropatlowmagnificationunderthemicroscope,useanacupunctureneedleorothersharpobjecttobreakthecrystal.Saltcrystalsareextremelyhardandcanevenbeheardtosnapuponbreaking.Mostproteincrystalscrumbleandsmasheasilyuponprobingthem.
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3.IZITdyefromHamptonResearchsellsaproprietarybluedye,IZIT*,whichbindstomostproteins.Thedyewillconcentrateinthecrystalsiftheyareproteinandturnthemblue,althoughtherearereportsof“false-negatives,”i.e.,proteincrystalsthatfailedtoturnblue.TheIZITdyeitselfcanalsocrystallize,usuallyappearingasextremelylongthinneedlesthatformwithinminutes.Instructionsforusingthedyeaccompanytheproduct.*Izitdyeis0.5%methyleneblue.4.InherentUVfluorescenceoftryptophansThismethodrequiresthatyouhaveaUVimager.
UV check
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WednesdayExperiment6:HowtocomparesaltandproteincrystalsbythesnapmethodandIZITdye(methyleneblue).Purposeofthisexperiment:Oftenpeoplelookintheirdropsandwonderifthecrystaltheyareseeingisproteinorsalt.TheobvioustestistoputitintheX-raybeam,butsometimesthecrystalsaretoosmalltobemounted.Beforeoptimizingtomakelargercrystalsitcanbeusefultoknowifitisworthpursuingatall.Onewayistosnapthecrystals,anotherwayistouseadye.Herewewillusethe“snap”methodandtheIZITdye.Materialsrequired:
• Lysozymecrystals,preparedinadvance.• onemicrobatchTerasakidish,filledwith5-8mlparaffinoil• a50mlFalcontubewithsaturatedNaCl(forthesaltcrystals)• needles(acupuncture,sewing,orhypodermicneedles)forthesnaptest• IZITdyefromHamptonResearch(=0.05%methyleneblue)
Today'sexperiment
• FillaTerasakiplatewithparaffinoil.TransferafewNaClcrystalsfromthesaturatedsolutiontotheplate.
• TakeaneedleandbreakaNaClcrystal,thenoneofthelysozymecrystals.NaClwill"snap".Saltcrystalsareveryhard.Convinceyourselfofthedifferenceinfeelbetweensmashingproteincrystalsandsnappingsaltcrystals.
• Repeatwiththelysozymecrystals.Noticethedifferencein“feel”.Proteincrystalsareverysoft,duetotheirhighsolventcontent.
• Instructionsfortestingthecrystalsfordyeabsorption:(copiedfromHampton'sbrochure)
"Place1microliterofIzitintothedropi.e.notthereservoir.(1microliterIZITper10microliterdropissufficient,mycomment).Protein,peptide,andnucleicacidcrystalswillabsorbIzitwithin1to4hours,takingonabluecolor.Thebluecolorwillintensifywithinthecrystalovertime,becomingadarkerbluethanthesolvent.PrecipitateandinorganiccrystalswillnotabsorbtheIzitandwillnotbecomeblue.Ifthebluebackgroundofthemotherliquoristoodark,simplydiluteIzit1:10or1:100withwaterandrepeat."
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ThursdayExercise7.Ligandsoaking(eosinred)intopre-grownlysozymecrystals.Materialsrequired: Dishwithpre-grownlysozymecrystals. EosinRed,powderora1%solutionProcedure: Carefullyaddtheligandtothedrop.Inthefigurebelow,theeosinredprecipitatedheavily,butthedyehasstillpenetratedthelysozymecrystal,turningitred.Figurebelow.Acrystalsoakingexperiment.Thiscrystaloflysozyme,0.3x0.3x0.3mm,hasbeengrownbyvapordiffusion.Thedropletvolume(afterequilibrationagainstthereservoirsolution)isapproximatelyone-halfoftheinitialvolumeof4microliters.Avolume(0.2microliters)ofareddye(1-10%Eosinscarlet)hasbeencarefullypipettedintothedroplet.Duetoitslimitedsolubilityintheconstituentsofthedroplet,muchofthedyehasprecipitated,butsomeofitstillappearstohavediffusedintothecrystal.Afterabout4h,thecrystalhadturnedcompletelyred.
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Thursday
Exercise8.CBH1Ligandsoakingbythreemethods
Materialsandsolutions• Pipettes,downto1ulvolume• soakingsolvent=precipitantsolutionwith20%mPEG5K,50mMNa-MESbuffer
pH6.0,10mMcobaltchloride,and12.5%glycerol(provided)• crystallizationdropswithniceCBH1crystals,yourownorpregrown• ligandpowder(s)–tobedecided• ligandsoaksolution(s)–ligandstobedecided,dissolvedinsoakingsolvent• smallspatula• acupunctureneedles• loopsmountedonbase,forpickingupandtransfercrystals• magneticwandChoosecrystalsforsoaking
1. ExamineyourCBH1crystallizationplateunderthemicroscope.Noteif/wheretherearecrystals,orprecipitate,orifthedropsareclear.
2. Selectsomecrystallizationdropswithnice-looking,singlecrystal(s)thatmaybeusedfordatacollection,amongyourown,orpregrowncrystalsprovidedbytheteacher(s).
Method1:Ligandpowdersoak
3. Takeoffthecapwiththeselectedcrystallizationdrop,turnupsidedown,andplaceinfrontofyou.
4. Takeaslittleaspossibleofligandpowderonthetipofaspatula(orsimilar),andplacethepowderattheedgeofthedrop,orneartheedgeofthedrop.Inthelattercase,useaneedletomakepowderanddropcomeincontact.
5. Putbackthescrewcapaboveitswell.Method2:Addligandsolutiontocrystals
6. Take10ulligandsoaksolutioninaneppendorftube.7. Takeoffcapwiththecrystallizationdroptobesoaked.Turnupsidedown
andplaceinfrontofyou.8. Take1ulligandsoaksolutionandplaceneartheedgeofthe
crystallizationdrop,butnottouching.9. Useaneedletodrawalineofliquid(i.e.,aliquidbridge)betweenthe
twodrops.Thetwodropswillbeconnectedandtheligandwillgentlydiffuseintothedropwiththecrystals.
10. Putbackthescrewcapaboveitswell.Tightengently.
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Method3:Transfercrystalstoligandsoaksolution
11. Pipet100-500ulofsoakingsolventasreservoirintwoemptywellsonaplate.
12. Placea2uldropofligandsoaksolutiononnewscrewcaps.Placeontopofthewellswiththesoakingreservoirs.Donottightenthecapatthisstage.
13. Putaloopofsuitablesizeonamagneticwand.14. Makesurethateverythingisreadyforcrystalpickingandtransfer.Takea
deepbreath,becausethefollowingstepsneedasteadyhandandneedtobedonequickly.
15. Takeonescrewcapwithcrystalstobesoakedandplaceitunderthemicroscope.
16. Placeonecapwithligandsoakingdropclosetotheonewithcrystals.17. Lookthroughthemicroscope.Immersetheloopinthedrop.Placethe
looparoundonecrystal.Dragtheloopout,andhopefullythecrystalfollows.
18. Transfertheloopcontainingthecrystalintotheligandsoakingdrop.19. Returnthecapsabovetheirrespectivewells.20. Rinsetheloopinabeakerofwater.Letitdryinair.21. Lookattheligandsoakingdropunderthemicroscopetoseeifyou
managedtotransferthecrystal.22. Repeattheprocedure,usinganothercrystaldrop,andalternating
betweenthesoakingdrops,togivethemsometimetoequilibrate/recoverabovetheirreservoirsbetweenexposurestoair.
23. Takeaquicklookatallyoursoaks,beforeyouputthemawayuntiltomorrow.
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Thursday
Experiment9.Cryocoolingofcrystals
PracticeSession
Part1:Readthissafetywarningfirst!LiquidNitrogenDemonstrationsSafetyNotes&ConcernsSOURCE:http://webs.wichita.edu/facsme/nitro/safe.htm
Liquidnitrogenisadangerousmaterial.ThefollowingisanexcerptfromtheAirProductsNitrogenMaterialSafetyDataSheet:
Abackoftheenvelopecalculationindicatesthattheentirecontentsofa10Literdewarbeingspilledinaunventilated274squarefootroomwithan8footceilingwouldreduceoxygenlevelsbelowthe19.5%levelwhereAirProductsrecommendstheuseofarespirator.Sincemostclassroomsarelargerthanthis,suffocationdoesnotrepresentamajordanger.Whentransportingtheliquidinacar,however,itisprobablyagoodideatoopenawindow.
Thepossibilityoffreezeburnsrepresentsamuchmoreseriousdangerandisthereforeourfirstconcern.Thisdoesnotmeanthatthedemonstrationitselfisdangerous,butitdoesmeanyoumustbecareful.Dangersinclude:
• Nitrogencanspatter(possiblyineyes)whilebeingpoured.• Flyingchunksoffrozenobjectscouldcauseeyeinjury.• Studentsmightreachoutandtouchnitrogenorothercoldobjects.As
mentionedabove,contactwithnitrogencancausetissuedamage,andthismustbeprevented.
Thereforespecificsafetyprecautionsshouldinclude:
• Teachersmuststresstotheirstudentstheimportanceofnottouchingfrozenobjectsornitrogen.
• Weargoggleswheneverpouringordumpingnitrogen.Nitrogencanspatterintotheeyes,andpotentiallyblindingpiecesoffrozenthingscanflyaroundwhenwedropit.
• Useagloveand/ortongstohandleanyobjectgoingintooroutofnitrogenandtocarrythenitrogendewar.
Ifcryogenicliquidorcoldboiloffcontactsaworker'sskinoreyes,frozentissuesshouldbefloodedorsoakedwithtepidwater(41-46C).DONOTUSEHOTWATER.Cryogenicburnswhichresultinblisteringordeepertissuefreezingshouldbeseenpromptlybyaphysician.
ENDOFCOPIEDMATERIALFROMhttp://webs.wichita.edu/facsme/nitro/safe.htm
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Nowarmwateravailable?Putthefrozentissue(usuallyyourfingers)underyourarmpit,skin-to-skintowarmit.
Thefigurebelowshowsafrostbiteaccidentfromourownlab.
Part2:Equipmentrequireda. Magnetictongsb. crystalcaps,vials,andloopsc. foamdeward. liquidnitrogene. glycerolandmotherliquorsolutionsf. safetygoggleswhenpouringtheliquidnitrogeng. clothglovescoveredwithvinyl/latexglovesh. microscopesi. pipettes
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Part3:MethodforcryocoolingwithpucksExampleforlysozymecrystals.
• Selectthecrystalsyouwanttomount.Whatmotherliquordidtheygrowfrom?Iftheygrewinacryoreadysolution,youcanputthemintoLN2directly.
• Ifthemotherliquorisnotcryoready,thecrystalmustbetransferredtoorsoakedinacryosolution.Seeexampleforlysozymebelow.
• Preloadthepuckwithemptyvialsandplaceinafoamcontainer.Fillthefoamcontainerwithliquidnitrogen.SeeFig1aandb.WEARAPPROPRIATEEYEWEAR.
• Selectaloopsizeappropriateforthecrystalsize.SeeFig.2.Differentlooptypesareavailable.SeeFig.3.Trydifferentonestofindyourfavorite.
• PUTONYOURGLOVES.• Pickupthemagneticcapcontainingtheloopwiththemagnetictongs.• LoopthecrystalfromitsoriginaldropletSeeFig.4.Ifacryosolutionis
required,transferthecrystaltothecryodropfor1second.• Putthecrystalasquicklyaspossibleintothenitrogen.• Label/recordwhichcrystalisinwhichpuckandtheposition.Exampleforlysozymecryosolution
• Makea1mlsolutionof70%motherliquor:30%glycerolinaneppendorftube.Tip:Warmthe100%glycerolinaheatblocktomakeiteasiertopipette.
• Puta2-5microliterdropletofthecryosolutionnexttoyourdropletcontainingthecrystals.Thisisthe“cryodrop”.
• Loopthecrystalfromitsoriginaldropletandtransferthecrystaltothecryodropforabout1second.
• Afterthe1-secondcryosoakinthecryodrop,putthecrystalasquicklyaspossibleintothepre-cooledvialintheliquidnitrogen.
• Alternative:youcanalsoaddthecryosolution(1or2microliters)totheexistingdropthatcontainsthecrystals.
25
MountingthecrystalsinspinepucksFig1a.Beginbyfillingthepuckwithemptyvials.(Noliquidnitrogenyet).
Fig1b.Thenfillthefoamdewarwithliquidnitrogen.Keepthenitrogenfilledtothetopatalltimesforbettercryocooling.
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Figure2:Thecryoloopsizeshouldbeappropriateforthecrystal.FigurecourtesyofBergfors,T.,Ed.“ProteinCrystallization,2ndEdition”2009.
MiTeGenDualthicknessmicroloop
MiTeGenmeshloopsforsupersmallcrystals(wedonothaveinthislabexercise).
Hamptonnylonloop
MiTeGenDualthicknessmicromount
MolecularDimensionslitholoop
andvariantsthereof.
Figure3.Typesofcryoloops
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Figure4:Loopingthecrystaloutofthedrop.PicturecourtesyofElspethGarman,chapter9“ProteinCrystallization”2ndEdition,EditedbyT.Bergfors,2009.
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FridayExperiment10.CryocoolingofcrystalsfortransportationanddatacollectionPurposeofthisexperiment:Select,pickup,andcryocoolcrystalsinliquidnitrogen,andarrangeindewarfortransportationtothesynchrotronforX-raydatacollection.EachgroupwillgetoneSPINEpuckwithroomfor10loopshangingincryovials,andshallcryocoolandsaveatleasttwoCBH1andtwoLysozymecrystals,andthentrytofillthepuckwithfurthercrystals,astimeallows.
Materials• Crystallizationdropswithnice,singlecrystals,yourownorpregrownbyteachers• loopsmountedonbase,forpickingupcrystals• cryovialsforcrystallizationloops• magneticwand• liquidnitrogen(LN2)• cryopucks(SPINEpuckswillbeusedthistime)• insulatedcontainerforkeepingthesamplepuckimmersedunderLN2• dewarfortransportationofthecrystalstothesynchrotron• protectiongoggles(“skyddsglasögon”)• gloves+mittens(toprotectyourhandsfromLN2)Preparation
1. Examineyourcrystallizationplatesanddropsunderthemicroscope,andevaluateeachdrop.Makegoodnotessincethisisthefinaldocumentationofyourcrystallizationexperiments.
2. Selectsomecrystallizationdropswithnicelooking,singlecrystal(s)thatmaybeusedfordatacollection,amongyourownand/orcrystalsprovidedbytheteacher(s).
"Nothingburnslikethecold".
GeorgeR.R.Martin
29
3. FilloneSPINEpuckwithcryovialsandplaceonapucksupportinacryogenicfoamdewar(orsimilar).
4. Beforeproceedingwithliquidnitrogen,makesurethatyouareawareofallsafetyprecautionsandwearsafetygogglesandsuitablegloves(andsafeshoesincaseyouspillonyourfeet).
5. Carefullyfillliquidnitrogeninthedewartocoverthepuck.Topupwhenithasstoppedboilingandsettledsomewhat.Keepittoppedupwithnitrogen,asdiscussedinthelecture.Youwantthedewarfilledtothebrim.
6. Collectandorganizeallstuffaroundthemicroscopethatyouwillneedforthecryocooling,i.e.crystalplates,magneticwand,cryoloops,dewarwithpuck,andthelistwhereyounotewhichcrystalgoesfromwhichplatetowhichpuckposition.
Cryocoolcrystals
7. Putonyourgloves!8. Pickupaloopofsuitablesizewiththemagneticwand.Underthe
microscopeyoucancomparethesizeoftheloopwiththecrystalthatyouwanttotake.
9. CBH1crystalsarealreadycryoprotected(with12.5%glycerol)andcanbecryocooleddirectly,withouticecrystalformation.
10. Takeadeepbreath,andbereadytoworkswiftlyandwithsteadyhands.11. Placethecrystaldropunderthemicroscope.Loopupthecrystal,transfer
promptlyintotheliquidnitrogen,andplaceinacryovialinthepuck.Pressthebuttononthewand,toreleasethecapwithloopandleaveinthecryovial.NB!Becarefultokeeptheloopwithcrystalunderthesurfaceonceitisimmersedinliquidnitrogen.
12. Putbackthecrystaldropimmediatelyaboveitsreservoir.Donotletthedropdryifithascrystalsleftthatmaybeused.
13. Noteinthelistforthepuckpositionwhatcrystalisinthevial,i.e.fromwhichplateandwhichposition/droplet,andfurtherdetailsandobservationsregardingthecrystal,e.g.size,shape,cracks,cocrystallizedwithligand,ligandsoak,etc.Formisonp.30.
14. Pickandcryocoolfurthercrystalstotryandfillyourpuck,astimeallows.15. NB!Lysozymecrystalsmustbesoakedincryoprotectantbefore
crycooling,asdescribedabove,onp.24.16. Makesurethatthetransportationdewarisfilledwithliquidnitrogen,and
thatapuckholderisinplaceinthedewarandiscooleddownandreadyforuse.
17. Transferthepucktothepuckholderandplaceinthedewar.NB!ItisveryimportantthatthepucksareintherightorderalreadywhenwepackthedewarhereinUppsala,sowecantakethemoutintherightorderwhenweshallcollectX-raydiffractiondataattheBioMAXbeamline.
18. Checkthatthelistofcrystalsinyourpuckiscomplete,andifnot,addthemissinginformation.
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PuckSystem:labelwhat'swhereDEWAR_____________________1black
TOP
12345
678910
2black
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678910
3black
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678910
4black
12345
678910
5blackBOTTOM
12345
678910
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PuckSystem:labelwhat'swhereDEWAR_____________________1red
TOP
12345
678910
2red
12345
678910
3red
12345
678910
4red
12345
678910
5redBOTTOM
12345
678910
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SummarizeyourplateresultsMaterialsrequired:MicroscopeYourcrystallizationplatesExercise:Lookatthedropsandtrytoidentifythedifferentphenomenaas:Clear,precipitate,denaturedprecipitateC,P,DPCrystals,crystalline,otherX,XN,OYoucanwriteyourobservationshere:Proteinorplatenumber: Method:Vapor-diffusion Hanging Sitting A1 A2 A3 A4 A5 A6
B1 B2 B3 B4 B5 B6
C1 C2 C3 C4 C5 C6
D1 D2 D3 D4 D5 D6
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Proteinorplatenumber: SCREEN:Method:Vapor-diffusion Hanging Sitting A1 A2 A3 A4 A5 A6
B1 B2 B3 B4 B5 B6
C1 C2 C3 C4 C5 C6
D1 D2 D3 D4 D5 D6
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Proteinorplatenumber: SCREEN:Method:Vapor-diffusion Hanging Sitting A1 A2 A3 A4 A5 A6
B1 B2 B3 B4 B5 B6
C1 C2 C3 C4 C5 C6
D1 D2 D3 D4 D5 D6
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Appendix1:ReducingAgentsinCrystallizationTrialsWhentousethem:— Ifyourproteinhasfreecysteines— TopreventoxidationofSe-methionine-substitutedproteins— IfyouarenotSUREyourproteinhasfreecysteines,add1mMbeta-
mercaptoethanol,theweakestofthesereducingagentsHowmuchtouse?1. Example:1mMproteinsolution(say,20kDproteinat20mg/mL)with6free
cysteinesneeds6mMDTT.2. Thelargetetramergalactosidaserequired70-140mMBMEinthecrystallization
conditions.Whichonetouse?YoucanchecktheDLSprofileoftheproteinwiththedifferentreducingagents.Itmaybenecessarytotrydifferentconcentrationstofindtheoptimumone.Allofthesearemorestableat4than25degreesC,butallofthemwillautooxidizeeventually.TheirabilitytoreduceexistingS-Sbondsisconcentrationdependent.Reducingagent
#SHgroups
Howlongdoesitlast?
Comments
betamercapto-ethanol
1 2-3days • Volatileandstinky(youcansubstituteMESNA,2-mercaptoethanesulfonate,ifyoudon’tlikethesmell).
• Addtothereservoir1microliterper1ml
DTT(oritsisomer,DTE)
2 3-7days;20%oxidizesafter18hratpH8-9
• Hardtorenewinhangingdrops,moreusefulindialysisbuttons.
• Don’tuseonNicolumns,itreducestheNiions.
• AllmetalsdegradeDTT,soinclude1mMEDTA.
• Auto-oxidizedDTTabsorbsat283nm.
TCEP=tris2-carboxyethylphosphine
0 3-4weeks • Verypowerfulanti-oxidant• Cannotbeusedwithphosphate• EDTAinactivatesit• CanbeusedonNi-IMACcolumns• WillevencleaveSHbridges,sodon’tuseit
ifyouhaveS-Sbridges• Itisveryacidic;adjustthepHofTCEPto
7.0(orbuyittitratedfromPierce).
36
Appendix2:CBH1informationWhyCBH1?• Wehaveampleamountsofprotein.• Wecanobtaincrystalsindays,i.e.withinthetimeframeofthecourse.• Crystalscanbegrownincryoprotectedconditionsandcanthusbedirectly
cryocooledinliquidnitrogen.• CBH1crystalsoftengivestrongdiffractiontohighresolution.• Itisamore“reallife”examplethanlysozyme,providingadditionalaspects:• Optimizedconditionsareneededtogetgoodcrystals.Itiseasilyseenhowthe
crystalformationisaffectedbychangesinionicstrength,buffer,pH,proteinconcentration,proteinbatch,precipitantconcentration,metalion,etc.
• Crystalsmaybebigandbeatiful,andyetgivecrappydiffraction,wellillustratingtheaxiomthat“Beautyisonlyskindeep!”.Thereforeyoumayhavetoscreenseveralcrystalsuntilfindingagoodone.
• Ithaspost-transcriptionalmodificationstolookforinthesolvedstructure.• TheN-terminalglutamineresidueiscyclizedtopyroglutamate(PCA).• Allcysteinesshouldformdisulfidebridges.• Theproteinisglycosylated,andyoumayseesugarattachedtotheproteinat
oneortwoofthethreeN-glycosylationsites(Asn-Xxx-Ser/Thr).• Adivalentmetalionisrequiredforcrystallization(I222form).Co2+seemstobe
best.Ni2+,Ca2+,Mn2+worktoo,butgivelessnicecrystals.• Onemetalionsitsona2-foldsymmetryaxis,whichisofinterestbecauseit
requiresspecialtreatmentinstructurerefinement.• Theactivesiteiseasilyseeninthestructure,intheformofacellulosebinding
tunnelthatrunsalongthewholecatalyticdomain.• Variousligandscanbindattheactivesite.• Wehavecatalyticmutantsandcanthussoakinnaturalsubstrates.QuickfactsFullengthCBH1:497aa,~56kDa,pI~3.7;Catalyticdomain(CD):439aa,~47kDaUsualspacegroupI222;unitcell82-84x82-84x109-111Å(α=β=γ=90°)Glycosidehydrolasefamily7(GH7).Retaining.Hydrolyzesβ-1,4-glycosidicbondswithretentionofanomericconfiguration(β →β),viacovalentglycosyl-enzymeintermediate.CatalyticnucleophileGlu212;catalyticacid/baseGlu217.BackgroundCBH1standsforCellobiohydrolase1.ItisalsoknownasCBHI,andCel7Abecauseitbelongstoglycosidehydrolasefamily7(GH7)intheCAZYdatabase(CarbohydrateActiveenZYmes).Itisthemajorcellulose-degradingenzymeproducedbytheascomycetefungusTrichodermareesei(alsoknownasHypocreajecorina).CBH1consitutesnearlyhalfofthetotalamountofsecretedproteinwhenthefungusisgrownoncelluloseascarbonsource.ThenativeCBH1enzymeisasinglepolypeptidechainof497aminoacidresidues.Itconsistoftwoindependentlyfoldedmodules:anN-terminalcatalyticdomain(CD)of~430residues,andaC-terminalcellulosebindingmodule(CBM1)of~35residues,connectedbyanextended,unfoldedandflexiblelinkerpeptideof~30residues.ThestructureoftheCBMhasbeendeterminedby
37
NMR(Kraulisetal,1989,Biochemistry28:7241-57).NoonehasyetmanagedtocrystallizethecompletefulllengthCBH1enzymewithbothmodules.However,byusingtheproteasepapainitispossibletocleaveoffthelinker-CBMtail,andtocrystallizetheisolatedcatalyticdomain(residues1-439).Thefirststructurewaspublishedin1994(Divneetal,1994,Science265:524–8).Todaythereareover25structureentriesinthePDB,includingseveralmutantsandvariousligandcomplexes.MostofthemarefromcrystalsofspacegroupI222withunitcelldimensionsaround83x84x110Å(α=β=γ=90°).HeterologousexpressionofCBH1inE.colihasnotoriouslyfailedtoproduceeithersolubleoractiveenzyme.Inyeast,expressionlevelsarelow,andthepropertiesofexpressedCel7shavebeenunpredictable,probablyasaresultofboththelackofN-terminalglutaminecyclizationofCel7Aexpressedinyeastandthesignificantvariationinproteinglycosylation(Danaetal,2014,BiotechnolBioeng111:842–847).MostCBH1mutantshavebeenmadebyhomologousexpressioninT.reesei.CatalyticallydeficientmutantsofCBH1,madebyisostericmutationofthecatalyticnucleophileGlu212orthecatalyticacid/baseGlu217toglutamine,havebeeninstrumentalforthestructure-functionstudies(Ståhlbergetal,1996,JMolBiol264:337-349).TheE212QandE217Qmutantshaveenabledthedeterminationofligandcomplexstructureswithnaturalsubstratesboundattheactivesitewithoutbeinghydrolyzed(Divneetal,1998,JMolBiol275:309-325;Knottetal,2014,JAmChemSoc136:321-329).
Figure5:TrichodermareeseiCBH1isaprocessivecellulase.Left:Acellulosechainisthreadedintothesubstratebindingtunnel.Tryptophansidechainsformhydrophobicsugarbindingplatforms,Trp40atsubsite-7andTrp38atsubsite-4.Thecleavagesitebetweensubsites-1and+1isoutsidetheviewofthisimage.Right:SnapshotfromanMDsimulationofglycosylated,fullengthCBH1,withlinkerandCBM,bindingtoacellulosechainonthesurfaceofaplantcelluloseImicrofibril.Theproteinandcellulosearerenderedassurfacemodelsandtheproteinispartlytransparenttoshowthecellulosechainboundintheactivesitetunnel.Aminoacidresiduesaregrey,N-linkedsugarsareblue,O-linkedsugarsareyellow,andthecelluloseisgreen.From:Payneetal,2013,PNAS110:14646-51.
38
Uppsala University Uppsala is Sweden’s fourth largest city and one of its oldest. Ever since the Viking Age it has been an important cultural centre. Uppsala is a rapidly growing city and is becoming increasingly integrated with the wider Stockholm region, Sweden’s most dynamic growth region. At the same time, Uppsala has kept many of its small-town characteristics. Offerings are myriad, but everything is close by – often within convenient cycling distance. The compact city centre with the cathedral, the River Fyris, and several small squares, parks, cafés, restaurants, and historic buildings lend the city its character. Did you know? Uppsala University has several museums. Large collections of art, science history and cultural history have been tended in Uppsala since the Middle Ages.
Uppsala has a rich musical scene on offer to both audience and musicians.
The Royal Academic Orchestra was founded in 1627.
The Botanical Garden is home to thousands of plants from the Scandinavian mountains to dry deserts and drenched rainforests.
The Silver Bible was donated to Uppsala University in 1669.
39
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Our university produces world-class research in several fields. Our degree programmes lead to important jobs, and our knowledge is sought-after by industry and society as a whole. We act locally and globally for asustainable, thriving and better world.
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Natural resourcesin focus
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