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第三章蛋白质晶体学1.蛋白质的结晶和晶体生长[主要内容]：蛋白质结晶的原理、技术和方法、对材料的要求、晶体生长的理化条件、晶体的鉴定。2.晶体学基本概念3.衍射数据的收集[主要内容]：晶体的收集与冷冻处理；X射线源的选择；衍射线记录装置及其使用方法；衍射数据收集的全自动化。4.蛋白质结晶结构的解析[主要内容]：蛋白质晶体空间群(Spacegroup);相位(Phase)的测定；电子密度图的解释；结构模型的修正与精化。5.pdb数据库文件以及结构的三维显示RapidGrowthofX-rayCrystallography1.RecombinantDNA2.MorepowerfulX-rays=Smallercrystals3.Automateddetectors4.Fastcheapcomputers5.Bettercomputingalgorithmsandprogramsuites6.Moresystematicmethodsforgrowingcrystals7.Crystalcryopreservation8.NewapproachestothephaseproblemCrystallizationofProteinSectionOneMakecrystalsofyourprotein90.1-1.0mminsize9Proteinsmustbeinanordered,repeatingpattern.Proteincrystallographyworkflowproteincrystalstructuresolutiondatacollectionexpression&purificationphasingcrystallizationGeneralReferences:1.PreparationandAnalysisofProteinCrystals,A.McPherson(RobertE.KriegerCo,1989)2.PrinciplesofProteinX-RayCrystallography,J.Drenth(Springer,1999)3.MacromolecularCrystallography(PartA,B,CandD)MethodsinEnzymologyVol.276/277/368/374.(AcademicPress,1997and2003)WebSites:1.CrystalGrowing101::::://:::(镜头)(crystal)Crystallographer&ComputersElectronDensity(Structure)X射线的波长与C－C键长一个数量级为什么选择X射线？只能观察到比波长大的物质高真空•WhyX-rays?–CuKaWavelength,1.5418Å–verysimilartodistancebetweenbondedcarbonatomsPrincipletheuseofelectromagneticradiationtovisualiseobjectsrequirestheradiationtohaveawavelengthcomparabletothesmallestfeaturesthatyouwishtoresolve.WeoftenuseX-raysemittedfromcoppertargetsbombardedwithhighenergyelectrons,whichemitatseveralcharacteristicwavelengths:theoneweuseiscalledCuKa,whichhasawavelengthof1.5418Å.Thisisverysimilartothedistancebetweenbondedcarbonatoms,soitiswellsuitedtothestudyofmolecularstructure.•Whyelectrondensity?charge/mass（e/m）:electronsatomicnucleiorprotons(质子)electronsacceleration⇒electromagneticradiationPrinciple•Whatweseeastheresultofacrystallographicexperimentisnotreallyapictureoftheatoms,butamapofthedistributionofelectronsinthemolecule,i.e.anelectrondensitymap.F(s)=∫Vσρ(x)e2πix.sdxX-raycrystallography-elasticscattering:same,altereddirectionεt:EmissioncoefficientThomsonscatteringChargetomassratioofelectronisabout2000timesastheelectronofatomicnucleiorevenprotons.Intensityofscatteredradiationisproportionaltothesquareofthecharge/massInelasticscattering,excitinganinner-shellelectrontoahigherenergylevel,isusefulforprobingsuchexcitationsofmatter,butnotindeterminingthedistributionofscattererswithinthematter•Whycrystals?–Hugenumberofmoleculesinsameorientation•X-rayscattering(散射)fromasinglemoleculewouldbeunimaginablyweakandcouldneverbedetectedabovethenoiselevel,whichwouldincludescatteringfromairandwater.•Acrystalarrangeshugenumbersofmoleculesinthesameorientation,sothatscatteredwavescanaddupinphaseandraisethesignaltoameasurablelevel.Inasense,acrystalactsasanamplifier.•Ifthewavesaddupinphaseinsomedirections,theyhavetocanceloutinalotofotherdirections.Thatiswhythediffractionpatternfromacrystalisanarrayofspots.Principle•Thereareanumberofpotentialbottlenecksindeterminingacrystalstructure,butgrowingausefulcrystalcanbethemostseriousone.•Ifyoucan'tcollectanydataoronlybaddata,youwon'tbeabletosolveastructure.CrystallizationGrowthofcrystalswithgooddiffractionquality:amajorhurdleinproteincrystallography.¾Stilllikeanart,manystories(蛋白质结晶与其说是一门科学，倒不如说更像是一门艺术!)¾Progressinunderstandingthebasicmechanismsofcrystallizationusingphysicalapproaches¾Trialanderror:systematiccrystalscreening¾Commercialscreeningkits¾Factors:concentration(10-50mg/ml),precipitates,pH,temperature,detergents,additives,metals,etc.蛋白质结晶并不是玄学也不是变魔术，而是一门科学性很强的操作艺术，是存在可遵循的科学规律的。关于蛋白质结晶，尽管人们已经积累了大量的经验，但仍然缺乏有效的理论指导,从某些角度看起来跟艺术类似。仅限于找生长晶体条件的过程待结晶的蛋白质分子或复合体蛋白质晶体内部结构为三维周期有序重复排列，要求每个结晶重复单位(分子或其复合体)的化学组成与分子构象是均一的。OOZZXXYY蛋白结晶母液完全无序状态高度有序状态不可能自发地完成！必须在某种外界的“力”的帮助下才可能完成这种熵减的过程。清澈的蛋白质溶液↓饱和溶液↓过饱和溶液↓发生沉淀↓蛋白质的无定形沉淀施加引起蛋白质溶液饱和度变化的因素当满足“适当的”条件，蛋白质就有可能以晶体形式从溶液中析出。蛋白质溶液从清澈到浑浊的动态过程SupersaturationToaddmoreofasubstance(toasolution)thancannormallybedissolved.Thisisathermodynamicallyunstablestate,achievedmostofteninproteincrystallographybyvapordiffusionorslowevaporationtechniques.Zone1-Metastablezone.Thesolutionmaynotnucleateforalongtimebutthiszonewillsustaingrowth.Itisfrequentlynecessarytoaddaseedcrystal.Zone2-Nucleationzone.Proteincrystalsnucleateandgrow.Zone3-Precipitationzone.Proteinsdonotnucleatebutprecipitateoutofsolution.盼望已久！很窄的范围ΔG=ΔH-TΔS因此:ΔH0结晶条件为了获得某种蛋白的结晶，往往需要进行一些适当的预备实验摸索。1.蛋白的纯度一般来说，蛋白越纯，越容易获得结晶，长成单晶的可能性也越大。除个别情况外，一般蛋白纯度应达到90％以上。2.蛋白的浓度对大多数蛋白来说，蛋白质浓度在10~50mg/ml较好。一般来说,蛋白浓度越高，有利于分子间相互碰撞而聚合，但是蛋白浓度过高,往往形成沉淀;蛋白浓度过低，不易形成晶核。3.晶种有些不易结晶的蛋白，需加入微量的晶种才能形成结晶。在加入晶种前，要将溶液调整到适于结晶条件下，加入的晶种开始溶解，还要追加沉淀剂。直到晶种不溶解为止。当达到