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SharingandimprovingtheBenchexperiencebyChenGuman
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Crystallographyforbeginnerspart5monitoringandevaluating
crystallizationexperimentsresults
PostedbyChenGumanon13/04/2013inCrystallography,Protein,Proteincrystallization,Tips
So,youvesetyourrstplate(s)asIhavedetailedinmypreviouspostaboutseingupcrystallizationplates
(hps://benchwise.wordpress.com/2012/11/17/crystallographyforbeginnerspart4seingupyourrstcrystallizationexperiment/).
Thatsgreat,yetthisisonlythersthalfofacrystallizationexperiment.Nowyouneedtomonitorforcrystalgrowthandinterpretthe
resultofthecrystallizationexperimentssoyouknowwhichfutureexperimenttosetuptoobtaincrystals(unlessyoureluckyandalready
obtainedacrystalintheinitialexperiment).Itmightsoundlikethefunpartyetthisstepiscrucialforobtainingcrystalswhenthosedont
comesoeasily(whichisthecaseformostproteins).
Whatdoyouneed?
Sinceproteincrystalsarepreysmall,rangingfrom0.001mmtoapproximately0.1mm,youwillneedastereomicroscopetoobserve
them.Whileitmightseemexcitingtolookforproteincrystals,thisisatediouswork,scanninghundredsofdropsforcrystalgrowth.
Today,withtheintroductionofroboticstothelifescienceapplications,therearenumerousroboticsforimagineofproteincrystalthatcan
makethelifeofacrystallographermorecomfortable.However,sincemanycrystallographylabsdonthavesuchrobots,youwillmost
probablydothismanually.Takeintoconsiderationthatthisworkisalsotimeconsumingsinceyoucanquicklyaccumulatedozensof
platethatneedtobemonitoredonaregularbasisjustbyusingseveralcrystallizationscreenswithcoupleofconcentrationsandat
dierenttemperaturesYoullneedalsoapenandapapertomarkyourselfthenameoftheplate,positionofthehitsandtheir
crystal/precipitatemorphology.
Whenshouldyoumonitoryourplates?
YoullbemostcuriousandeagertolookatyourplaterightawayandyouSHOULDdojustthat.Lookingattheplateatroughlytime0
willgiveyoutheassessmentofthestartingpointofthisdynamicandcomplexprocess.Youshouldmonitortheplate(s)eachdayforthe
rstweekandthenchecktheplateonceortwiceperweekuptotwomonths,afterwhichyoushouldmonitoryourplatesonceortwicea
month.Thebestwaytomonitorthechangesovertimeisbytakingphotosandobservingthechangeovertime.Thisofcourserequires
accesstoastereomicroscopecoupledtoadigitalcameraandistimedemanding.Thus,unlessyouhaveaccesstoanimagingrobot(such
asthisone(hp://www.formulatrix.com/products/proteincrystallographytools/rockimager/index.html))youshoulduseascoring
schemesuchasbasedonnumbers(seepicturesbelow)coupledtoanexcelsheet128or64matrix(dependingonthetypeofplate):
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Theaboveplateisscoredaccordingtoinitials(C,cleardrop;PS,phaseseparation;P,precipitateetc.).Youcanalsoseetheexactdetailsof
everyexperimentalfactor(experimentationandmonitoringdate,proteinandcrystallizationagentconcentrations,temperatureandeven
theoriginofthespecicconditionstakenfromascreen).
Whatsinthedropandhowshouldyouproceed?
Inproteincrystallographyouraimistotransformtheliquidstateproteinunitsintoorderlyarrayedsolidstateproteinarray.Sincethe
processisverysensitivetonumerousenvironmentalaswellasinsolutionfactors,wewillexpecttowitnesseithernoaggregation(ifthe
saturationconditionsarenotmet)orseveraltypesofaggregates.Thetypeofaggregatewillgiveusclueswhetherweareonthecorrect
tracktowardproteincrystalgrowth.
Followingisapicturegallery(withasuggestednumberscoringscheme)whichrepresentsmostcasesofdropappearance:
Cleardrop(0)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iRHF29Mh/0/L/ClearL.jpg)
Thedropisclearwithoutanysignsofaggregations.Thismeansthateitherproteinorprecipitantconcentrationislow(orboth).
Contaminateddrop#1(Glass/ber)(0)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iVP4xf63/0/L/GlassL.jpg)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/i7dpZnFd/0/L/FiberL.jpg)
Thesearenonbiologicalcontaminatesthatenteredthedropwhiletheplatewasprepared.Glass(upperimage)canbeeasilymistakenfor2/8
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Thesearenonbiologicalcontaminatesthatenteredthedropwhiletheplatewasprepared.Glass(upperimage)canbeeasilymistakenfor
crystalsmonitoringtheseovertimewillprovetheiridentity.Fibersmightchangetheircolorunderpolarizedlightyettheircurvature
naturesignalthemasnoncrystalspecies.Notethatclosetotheberyoucanseetranslucentproteinprecipitates.
GranularPrecipitate(1)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/i5FNrTdx/0/L/Granular%20precipitateL.jpg)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iqSr6BBQ/0/L/Granular%20precipitate2L.jpg)
Theseareroundlikeprecipitatesthatusuallyformsmallclusters.Thebrownishkindareusuallydeadendsandevenafterseveralstepsof
optimizationinrarecasesthesewilleventuallyleadtocrystalformation.Translucentprecipitates,however,mightbeabeerstarting
positionforfurtheroptimization.
Fullprecipitate(2)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iPxbzdzZ/0/L/HeavyAg_microcryslL.jpg)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/ixJ5L83d/0/L/Full%20precipitateL.jpg)
Fullprecipitatesareusuallybrownishformsofaggregateswhichinmostcasesaredeadends.Inmanycasesthisphenotypeisduetothe
presenceoftoohighprotein/precipitantorofbothleadingtotheproteincrashingontolargeaggregates.Evenso,keeptrackoftheseas
someproteincrystalcanformoutofthesetoo(andbydoingso,clearingtheareasurroundingthecrystal).
Phaseseparation&oildrops(3)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iJgM3GGh/0/L/Phase%20separationL.jpg)
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(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/i57rd782/0/L/124504_11052010L.jpg)
Thesearehighlyconcentratedproteinaggregatesthatarepackedtogetheryetnotinacrystallaice,generatingroundshapes(upper
image)orgelatinousshapes(lowerimage).Thisisagoodplacetooptimizeconditions,especiallytemperature,sincetheprocessofcrystal
packingmightneedtobeslower/faster(andtemperaturecontrolsthat).Ifnothinghelps,youmightconsiderrecloningandchopo
possibletailsfromtheprotein.
Spherulites(5)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iG9QsfXH/0/L/SpherulitesL.jpg)
Thisisregardedasacertainformofmicrocystalline.Yes,thisareusuallyperfectlyroundandtheyshowthelightbefringingofcrystals.
SameasPhaseseparation,conductanexpansiongrid.
Microcyrstallineprecipitates(6)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iS6f9cgN/0/L/MIcrocrysL.jpg)
Thisisapromisingsignsuchhitsshouldbefurtheroptimized,inmostcasesrequiringtolowertheamountofnucleationevents,usually
byloweringtheproteinconcentration/precipitantorloweringtheincubationtemperature.
1Dcrystalsneedles(7)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/itFqV8BJ/0/L/NeedlesL.jpg)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/ihn92qst/0/L/Needles2L.jpg)
Needlesareonedimensionalgrowingcrystals,andassuchtheseareontheonehandverypromising,yetthereisaneedtoimprovethe
crystallizationgrowthdynamicssinceonlyonedimensionalisactivelyengagedinthegrowthprocess.Besidesplayingwithprotein
concentration/precipitate,pHandtemperature,youshouldalsovarythebuer.Ifyoundyoujustgetmoreandmoreneedles,withno
improvementtoward2Dor3Dcrystals,thenyoushouldconsiderrecloningandgeneratinganewproteinvariant.
2D&3Dcrystals(8&9)
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(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/i9RwbPTs/0/L/3Dcrystal_incompleteL.jpg)
(hp://www.cgp.co.il/Documentary/Benchwise/Dropmorph/iQTfCSBv/0/L/Rs14L.jpg)
Thesearethemostpromisingandthesoughtforproteincrystals.Sinceproteinunitsarepackedin2Dand3Ddimensionsthesecanlead
tohighresolutiondiractiondata.Asarule,acrystallographershouldaimatgrowingsinglelargecrystals.Lookingattheupperimage,
twophenotypicelementsanbeobserved:shapeofthecrystalsandtheirnumber.Undersuboptimalconditionsthe3Dcrystalswilllook
partroundasnotallcrystalsfacetswillbesharp,astrongindicationthateitherthegrowthrateistoofastorthattheproteinconcentration
istoohigh.Thelargenumberofcrystalunitsisaclassicdemonstrationofovernucleationpossiblyduetohighproteinconcentrationin
thedrop.Whentheamountofproteinissmaller,lessnucleiformandthusleadstofeweryetlargerproteincrystals.Evenso,when
obtainingmanysmallcrystals(whichhasclearsharpfacets)itisworthtochallengethemunderastrongradiationsourcesucha
synchrotronbeamsometimesthatsthebestyouget,andamongthesesomemightdiractbeyond2.5A.
Oneimportantnoteacrystallographyexperimentisinprogressuntilthedropdriesup.
Saltcrystals?!
Yep,saltscanquiteeasilycrystallizeandproducesomeverybeautiful(anduseless!)crystalsinyourdrop.Sinceyouarea
proteincrystallography(Ipresume),thesewillbeanuisanceinyourpathtogrowingproteincrystals.Howcanyoudierentiatethesalt
fromtheproteincrystals?herearesometips:
Saltcrystalsarehardanddense,thusyouwillmostlyseethemattheboomofthewell.ifyoutrytoprickthemwithaneedleyouwill
seethembouncingaroundhappily(thatsbecausesaltcrystalspackverywellandthusdoesnthavehighwatercontent).Protein
crystals,ontheotherhand,arecharacterizedbyarelativelylowerpackingdensity,whichislledbywatermolecules.This
characteristicsleadproteincrystalstobefoundattheupperpartsofthewell(lowdensity)andtobeveryfragile.Aglancingblowwith
aneedlewillusuallybesucienttomakethecrystalcrumblerightinfrontofyoureyes(thatsonegoodreasonwhyyoushouldnot
dothatifthisisyouronlycrystal).
Saltcrystalscanwithstanddehydration(shingthecrystalfromthewellandletitstayattheopenair).proteincrystals,ontheother
hand,willcrumble,crackanddisintegraterapidly.
Saltcrystalshaveaverystrongbingingpropertiesandusingapolarizerlteraachedtothemicroscopeyoucanseethestrong
colorfuleectofthepolarizinglight.Whileproteinscanalsobifringe,itisoflesserstrength.
UVabsorbanceifyourproteinabsorbsatUVwavelength(shouldcontaintheaminoacidstryptophanandtyrosine)thanunderUV
lightproteincrystalsshouldshineverybrightlywhilesaltcrystalswilllooklikeghostlyshapes.Forthisyouwillneedanimagerobot
thatisalsoequiptwithaUVlamp.
DyeSpecialdyes(suchasIzit(hp://hamptonresearch.com/product_detail.aspx?cid=4&sid=41&pid=33))willpenetratethecrystals
(sinceitislooselypacked)andbindtotheproteinsaminoacids,leadtocoloringofthecrystals.
Diractionthisistheultimatetestandifyouhavenextdoorahomesourcewithliquidnitrogenrunningthisshouldbetheonlytest
required.
(hp://en.wikipedia.org/wiki/File:Xray_diraction_paern_3clpro.jpg)
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(hp://www.cgp.co.il/Documentary/Benchwise/Misc/iWSw8SCT/0/L/diraction_Brushite1L.jpg)
TheaboveimageisadiractionimageofaSARSprotease,whilethelowerimagerepresentsadiractionpaernofaphosphatemineral,
Brushite(hp://en.wikipedia.org/wiki/Brushite).A(good)diractionpaernfromaproteincrystaliscommonlycharacterizedbymany
spotsacrossallresolutionlevels(low,midandhigh)whilethatofasaltcrystalisusuallysporadicandmostlyatthemidhighresolution.
Lowresolutiondiractiondataislocatedclosetothecenterofthediractionimage(closetothebeamstopblackcircle)whilethemidand
highresolutionextendstotheouterrimoftheimage,thehighresolutionlocatedattheedgeofthediractionimage.
Timetocelebrate?!Notsofast
Evenifyougotasingle,largeproteincrystal,dontpopoutthechampagnejustyet.Theultimategoalofaproteincrystalisnot
itsmorphologybutratheritspackinganddiractinginformation.Alargebeautifulcrystalcanbepoorlypackedandthusbothfragileand
willdiractpoorlyornotdiractatall.InfuturepostIwilldiscussthediractionexperiment,whatshouldyouexpectandhowto
proceedfromthere.
Nextpostwilldiscusshowonecanrescueadiculttocrystallizeproteindontmissit!
Dropphotos&crystalsarecourtesyoftheZarivachlaboratory(hp://lifeserv.bgu.ac.il/wb/zarivach/)andtheMacromolecularcrystallographyresearch
center(hp://lifeserv.bgu.ac.il/wb/crystalogy/),BGU.
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Tags:crystallizationexperiments,lifescienceapplications,proteincrystals,science Permalink
10commentsonCrystallographyforbeginnerspart5monitoringandevaluating
crystallizationexperimentsresults
thii
10/09/2013at08:54 Reply
beautifully.wrienariclemanythanksforyourniceexplainations.
ChenGuman
10/09/2013at09:15 Reply
HiThii
Thanksforyourfeedback!
Chen
QiangFan
18/09/2013at23:19 Reply
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18/09/2013at23:19 Reply
Itsapreygoodarticle,veryhelpfulandinformative.Thanks.
Qiang
Camille
19/02/2014at05:47 Reply
Juststartingsomecrystallographyexperiments,andveryexcited(andcurious)aboutit(Imacellularbiologywithnoknowledgeof
proteincrystallography)..andfoundyourexplanationsveryclear!Hopefullythecrystalwefoundtodayisnotsalt! Thanksfor
sharing.
penguin
05/03/2014at21:36 Reply
Veryinformativeindeed.thanks.
Daan
03/04/2014at10:52 Reply
Hello,
RegardingtheSaltandproteincrystalsIamalileconfused.
Ilearnedfromseveraldierentsourcesthatitisexactlytheoppositeofwhatyousay.
Saltcrystalsareveryfragileandeasytobreak,andyoucanevenhearadistinctclicksoundwhenyoubreakit.(whichiskindof
surprisingsinceitssuchatinytinycrystalbutthesoundisasloudasdroppingyourpenonthetable)
Andproteincrystalsareverystrongandhardtobreak.
PleasecorrectmeifImwrong
P.s.Whenwillpartsixbecontinued?
WithKindRegards
~Daan
ChenGuman
16/04/2014at07:35 Reply
HiDaan,
Saltcrystalsarehighlypackedspecies,withlilewatercontent,thusmakingthenverytough.Proteincrystals,ontheotherhand,
haveonaverage50%watercontentmakingthemfragileandsensitiveforhydrationlevels.
SeeHamptonResearchondistinguishingsaltandproteinhp://hamptonresearch.com/documents/growth_101/20.pdf
Perpart6well,thatwilltakesometime
Shweta
09/11/2015at23:09 Reply
ThankyousomuchChenforsuchaniceexplanation.Ihavedoneroboticscreeningjustaweekagoandlookingforwardformy
crystalstogrow.Iwasconfusedtoobservedmyplatesbutthisarticlemakeseasyforme.Thanksalotforsharingthis..
Joe
22/07/2016at07:17 Reply
Hi,
Nicenotesandphotos.
Justwantedtomentionthatyoumayconsiderchangingthatsbecausesaltcrystalspackverywellandthusdoesnthavelowwater
contenttothatsbecausesaltcrystalspackverywellandthusdoesnthavehighwatercontent.
ChenGuman
22/07/2016at20:16 Reply
HiJoe
ThankyouforthecommentIveappliedyoursuggestedchange.
Chen
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CreateafreewebsiteorblogatWordPress.com.
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