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

12345

678910

3black

12345

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

SVERIGES LANTBRUKSUNIVERSITET

SLU är universitetet som forskar och utbildar kring de biologiska naturresurserna, både på land och i vatten. Vi jobbar med hållbar ut-veckling av städer och landsbygd, och vi verkar för människors och djurs livskvalitet och välbefinnande.

Vårt universitet producerar forskning i världsklass inom flera områden. Våra utbildningar leder till relevanta jobb, och vår kompetens är efter-frågad i näringsliv och samhälle. Vi verkar både lokalt och globalt för en hållbar, levande och bättre värld.

SLU is a university where research and teaching are centred around biological natural resources, on land and in water, and how we can use them in a sustainable manner. Our work covers sustainable urban and rural development, as well as quality of life and well-being for both humans and animals.

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.

SWEDISH UNIVERSITY OF AGRICULTURAL SCIENCES

Med naturens resurseri fokus

Natural resourcesin focus

Did you know that…• SLU was founded in 1977, and celebrated 40 years as a university last year.

• SLU is the only Swedish university that trains foresters, landscape architects, veterinarians and agronomists.

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www.slu.se#sluinstallation

Visste du att…• SLU bildades 1977 och firade 40 år som universitet i fjol.

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