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Theproblem h,k,l,IandsigmaIhavebeencollectedinanXraydiffractionexperiment WehaveconvertedItoF,F=SQRT(I). Howdowegetcoordinatepositionsforthemacromolecularstructure?

experimental data
Fileofstructurefactoramplitudes

Structure PDBfile

Electrondensityequation(Densityrho(e/3)atx,y,zintheunitcell)

|F(hkl)|structurefactoramplitude(scalar)measuredinthediffractionexperiment. (phase)inradians.Thephaseofahklreflection.Lostduringthediffraction ( h )i di Th h f hkl fl ti L t d i th diff ti experiment Thus,F(hkl)isavectorwithamplitude|F(hkl)|andphase(hkl). hklarethemillerindices!!! xyzfractionalcoordinatesoftheunitcell.Samplingfrom01ineachdirection. electrondensity((xyz))iscalculatedbysummingallstructurefactorsateachxyz (samplinggrid)positionintheunitcell.Thus,ALLreflectionscontributetotheelectron densityateachplaceinthecell. Reducetheequationto1Dexampletolearnmoreabouttheequation.Homework elecdens.xls.

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Electrondensitywavesaredescribedbyvectors.2componentsamplitudeandphase

|Fhkl| =amplitudeofthewave =relativephaseofthewavecompared

tothesamewavewith=0 E g origin to the same wave with =0.E.g.origin If =0,densitywavewillalwayshave maximumatx=0

X=0

wavefreq. X=1 Onerotationaroundcircle=1unitdistinx. Shiftpositionofwave, relativetoorigin. h =ifh=1vectorrotates360in1xtrans. ifh=2vectorrotation720in1un

Theelectrondensity intheunitcellsofa crystalisaperiodicwavefunction.Itmay bedecomposedintoitscomponentwaves. Thesecomponentwavescorrespondto thefamilyofplanesdescribedby millerindicesh,k,l whichdescribetheorientationand which describe the orientation and frequencyofeachelectrondensitywave. hF Inthediffractionexperiment,we measureintensityofreflectionsat differenthkl.However,wehavelostthe relativephaseangles.Inthisfigure, A(F)ismeasuredatdifferentn(.e.gh). Wedonotknow ().Thisisthephase probleminXraycrystallography. IfweknowFand wecancalculate electrondensity. Sinceweknowbothletscalculatethe periodicwave..xlsfile

108 144 162 144 234

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Relationshipbetweenstructurefactor,h,k,lreciprocallatticeandelectrondensitywave k Reciprocallattice Witha*andb* perpendicularto aandb.Andthe h hkplane. Thereciprocal The reciprocal latticedistance Is1/d totheinter planarspacingin therealcell

http://www.ysbl.york.ac.uk/~cowtan/f ourier/fourier.html Vectordiagram Phase=0,e.g.maximumat origin

Realcellwith E denswave ingreenforthe h=1,k=0reflection Sinceh=1,planes cut1time. diffractionoccurs Offtheh=1planes

Thewaveextendsthroughoutthecrystal

Relationshipbetweenphaseandposition oftheelectrondensitywave

h=1,k=0,=0

h=1,k=0,=90

h=1,k=0,=180

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h=1,k=0,=0

h=2,k=0,=0

RelationshipbetweenXraydiffractionandelectrondensitywaves Electrondensitywaves 1. Frequenciesanddirectionsdeterminedbythemillerindices. 2. Amplitudesandphasesdeterminedbythepositionsandtypesofatomsintheunitcell

Xraywaves 1.Monochromatic(constant andconstantfreq. 2.Positionsofwavesonthedetectordeterminedbythecrystalsystem,unitcellconstants andmustsatisfyBraggslaw. 3. Diffractedbeamsexhibitdifferentphasesrelativetooneanother(hitdetectoratdifferent times). 4. Foragivenexperimentalsetup,thediffractedIntensitydependentonthepositionand 4 F i i t l t th diff t d I t it d d t th iti d typeofatomsintheunitcell. (Seethestructurefactorequation).

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FURTHERBREAKDOWNOFTHESTRUCTUREFACTORORANYVECTORintocomponentsA,B.

A=|F(hkl)|cos B=|F(hkl)|sin

=tan1(B/A)
|Fhkl|=SQRT(A2 +B2)

I=A2 +B2

If|F|=1 A=1,B=0 A=0.71,B=0.71

STRUCTUREFACTORsummationofscatteringvectorsofindividualatomsinunitcell F(hkl)correspondstoawavesscattered fromallatomsinthecrystalunitcellfora givensetofplanesh,k,l. f1,f2,andf3arethescattering contributionof3differentatomsin theunitcellofacrystal. Noteallatomscontributetothe measuredintensity.Thus,toeach structurefactor. Inthisfigure,atom1scatteringis greater2isgreaterthan3. Scatteringforeachatom brokendowninto b k d i A=f1cos andB=f1sin terms TheAandBcomponentsare Summedtogeneratethe structurefactorF= SQRT(A2+B2)andwithphase= Atan2(B,A),ortan1B/A

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StructureFactorequation(Summationoverallatomsintheunitcellforeachh,k,l.)

The structurefactorF(hkl)(vector)maybecalculatedifthex,yzpositionsofatomsare known. Fromx,y,z(fractionalcoordinates),(phase)inradianscanbecalculated. Theamplitudeofthestructurefactor|F(hkl)|isdefinedbytheatomicscattering factors (f)ofatomsinthecrystal hklarethemillerindices!!! EachF(hkl)consistsofscatteringcontributionsofallatomsintheunitcell. Conclusion:Todeterminethephasesofreflections,,youmustknowatleastone coordinatepositioninthecrystal.

TheIsotropicAtomicScatteringFactor 1. Resolutiondependent 2. Reducedbymovementoftheatom

Scatteringfactor(f)plotforoxygenvs.resolutionatB=0A2 andB=10A2

B=82<u>2

BfactorfoundinPDBfiles

IsotropicscatteringfactorsinelectronsfoundinInternationaltablesVol.4.(Table2.2) YourBfactorof10correspondstoameanatomdisplacementof0.36A

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H=1eat0.0 S=16eat0.0

5A

2.5A

1.7A

UNITCELLANDDIFFRACTIONPATTERNOFA1DCRYSTALLEARN2

B=10A2 foreachatom

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DIFFRACTIONDATAFORTHE1DCRYSTALLEARN2

RoleofthescatteringfactorinAlteringthestructurefactorcalculation

Isotropicf:6e 11.394.6210.41113.93 Resolutioncorrection7.523.456.69117.26 Resolution+Bfactorcorrection 5.042.384.48117.26

Thermalmotionofatoms resolution

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Structurefactorcalculationfortheh=8reflectionwithnoresolutionofBfactorcorrection:

Calculationofstructurefactorsfromatoms

2atoms6elec.each 1) x=0.1,y=0.16elec. 2) x=0.6,y=0.456elec Refertoscatfacttable. Note:greenedensitywave isacrossentirecrystal! y Calculatethestructure factorofthe2,0reflection. Donotapplyresolutionor Bcorrectionstothe scatteringfactorf

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Calculationofastructurefactorforthe

h=2,k=0,l=0reflection

f*

Whereisthe3,4onthe Reciprocallattice? CalculateFandphasefor theh=3,k=4reflection. Answer= F=11.4 F=11 4 Phi=126

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1,00reflection:Measuredbutabsentreflection SGdetermination

2atoms6elec.each 1) x=0.1,y=0.16elec. 2) x=0.6,y=0.456elec Lookbackatthe200

Thedistancebetweenatom1andatom2inthe Xdirectionis0.5.thus,the100wavecannot represent+densforbothatoms.Thisisthe basisfor systemicabsencesindiffractiondata whichareusedtodeterminethespacegroup

Comparedto100or300, largeFforh=4,k=0 reflection.

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HOWdoyoufindx,yzpositionofatomsincrystalfromjust|F(hkl)|data?

Noappropriatemodelstructureor structuralmodeltoodifferent

OftenheavyatomsdontbindORdontbindspecifically Heavyatomschangetheunitcell nonisomorphous

Generally,requireslabelingoftheprotein(SeMet).Greatifmolecule canbeproducedine.coli.Needanomolousscattere.gFeprotein

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ForSIRorMIRPhasingMethod

Fph

Fp

FPH =FP +FH Fh

DatasetofNative Crystalsderivatized Withheavyatom Hg,orU,orPb,orPt (lookatscattering factortable)

Datasetof Native crystal

Estimateof Heavyatom contribution

Measuretheintensity(amplitude)differencesbetweenHeavyatomdataset(Fph)and Measure the intensity (amplitude) differences between Heavy atom dataset (Fph) and Nativedataset(Fp). SubtractionofFph Fpisanestimateofthecontributionoftheheavyatomtothediffraction pattern. Findthephaseoftheheavyatom(Fh).Needtofindthexyzpositionoftheatom:PattersonMap.

FramesofdatashowingINTENSITYDIFFERENCEsbetweenNativeandderivatizedcrystal Nativecrystal derivativecrystal

Weakstrongweak

Spotintensities

Weakweakweak

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Pattersonmap allowstheidentificationofx,y,zpositionsofatomswithoutphases

Vectormapiszeroeverywhereexceptwhentheendsofthevectoru,v,w,areatomsx,y,z. Thesizeofthepeaksaretheproductoftheatomicnumbersforthetwo The size of the peaks are the product of the atomic numbers for the two atoms.2carbonspeakshouldbe6x6=36e Themapcontainsallselfvectors(e.g.atom1xyzatom1xyz=0,0,0).Thus,theoriginisthesum ofthesquaresoftheatomicnumbersofallatomsintheunitcell. example,atomsatx1andx2inthecrystalwillbelocatedatupositionsx1x2andx2x1 inthePattersonMap.Aswellasx1x1andx2x2. IfN atomsinaunitcellThePattersonmapwillcontainN2atoms

Bottomline:PattersonMapsprovideawaytodeterminex,y,zpositions withoutphases

NotedifferencesbetweenPattersonequationand

Electrondensityequation Electron density equation

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FeaturesofthePattersonmap u =x2x1,v=y2y1,w=z2z1 u =x1x1,v=y1y1, w=z1z1 u =x2x2,v=y2y2,w=z2z2 oru=0,0,0

4pattersonpeaks

u =x1x2,v=y1y2,w=z1z2

1. Alwayscentrosymmetric 2. Hasthesamelatticeastheoriginalspacegroup 3. Removealltranslationalcomponentsoftheoriginalspacegroupaddthecenter 4. TotalofN*NpeaksorN1*Ntodeterminenonoriginpeaks. Symmetry S t IfRealcellSG=P21

x,y,z x,y+1/2,z x,y,z x,y,z x,y,zx,y,z

ThenPattersonsymmetryP2/m

SpacegroupsymmetryhelpslocateatomsinPattersonMaps HARKERVECTORS SGsymmetryP2 Equivpositions 1. x,y,z 2. x,y,z

HARKERVECTORS

u=x x,v=yy,w=z z =u=2x,v=0,w=2z u=xx,v=yy,w=zz u=2x,v=0,w=2z

ORx=u/2andz=w/2y

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1DPattersonMapforC=0.1O=0.7FROMLEARN2data

1200 1000 800 600 Series1 400 200 0 0 -200 0.2 0.4 0.6 0.8 1

RemembertheseareeachVECTORS.weonlyhaveamplitudes

FPH =FP +FH

1.Ifyoudefinethelocationoftheheavy atom,youcancalculatethevectorFH. WithFHyoucandefinethephaseofthe protein(P).

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SIR

Twopossiblephasespossible withasinglederivative

FPH

FP FP FPH FH

Thepositionofaheavyatomhasbeendetermined.Thus,FHisavector. WewanttodeterminethephaseofFp

Using2derivativesresolvestheambiguityinthephasing 3intensitydatasets, 1. FP native(noheavyatom) 2. FPH1 heavyatom1 3. FPH2 heavyatom2

SIR

MIR

FH2

FH1

SIRmethodssometimeswork.Correctphasesareselectedusingamethodcalledsolvent leveling.E.g.Phasesmustbeconsistentwiththephysicalrulesofproteinstructuresandcrystals. Proteinsdensityispositive andcontinuousandsolvent channelsareconstantandlow.Starting estimatesof%solventistakenfromVm

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1DelectrondensitymapsFrom LEARN2DATASET A
140 120 100 80 60 40 20 0 0 -20 0.2 0.4 0.6 0.8 1

140 120 100 80 60 40 20 0 -20 0 0.2

0.4

0.6

0.8

1.2

A=ElectrondensitybasedonSFscalculatedwith atomsC=0.1,O=0.7 B=ElectrondensitybasedonSFscalculatedonly withatomO=0.7 C=PhasesfromBcalculation,withstructure factorsfromA. Partialandinaccuratephases,basedonlyonthe oxygenatom,revealthelocationofthecarbon inthee densitymap.

C
160 140 120 100 80 60 40 20 0 -20 0

0.2

0.4

0.6

0.8

c #1 #2 model Mo d c c c

c c c c

CollectanXraydiffractiondataset. CalculatestructurefactoramplitudesforKnownproteinstructure(Pmodel) However,dontknowwherethismodelisintheunitcell d k h hi d li i h i ll MolecularReplacement 1. FindsthecorrectrotationofPmodelrelativetoPcrystal(Pc). 2. FindsthecorrecttranslationofPmodelinPcrystal. 3. ThisiseffectivewhenPmodelisstructurallysimilartoPcrystal.Thebreakdown occurssomewherearound~35%orlesssequenceidentity.

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DifferencebetweenSIR/MIR(heavyatom)andSAD/MADanomalousphasingmethods

SIR/MIR

FPH =FP +FH

Fph

Fp

Fh

1.IntensitydifferencesbetweenFphandFp 2.Requiresdatacollectionon2crystals FriedelsLaw=Iof111=Iof111 |Fp(hkl)|(hkl)=|Fp(hkl)|,(hkl) SAD/MAD

Anomalous scatterer

BreakdowninFriedelsLawfor Anomalousscatteringatoms

1. CollectFriedelpairsofreflections,whichexhibitIntensitydifferences Fph+andFph (e.g.the111andthe111.Relatedby180 rotationofcrystal 2. ForSAD,requiresdatacollectionon1crystal..Perfectisomorphism. 3. ThisisgenerallySelenomethionine

AnomalousScattering

http://skuld.bmsc.washington.edu/scatter/

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Anomalousscattering(AS)causesa90 phaseshiftinthescatteringfactors.The sizeoftheASfactorcomponents(f(deltaf)andf)aredependentontheatom. AsaresultofAS,Freidelslawbreaksdownfortheanomalousscatter.Thisresultsin IntensitydifferencesbetweenFhklandF(hkl),whichcanbeusedtolocate Anomalousscatters(usingpattersonfunctions)andcalculatingFHfortheseatoms ResultinginphasesforFP

Vectordiagramshowingthechangein thescatteringfactor,whichcauses intensitydifferencesthatcanbeused Forphasing.

CollecttheSADdataattheabsorbtionedgeoftheanomalousscatter fandfplotforSelenium

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Friedelpairs

hklplane

BreakdowninFriedelslaw FPH(hkl)FPH(hkl) whenananomalous scatteringatomis present

FPH hklplane

FPH AttheappropriateXrayenergy,thewhiteatom Scatters anomalously and Friedel s law breaks down. This is ScattersanomalouslyandFriedelslawbreaksdown.Thisis becausethephaseoftheanomalousscattererisadvanced by90degrees(FIXED)relativetotheotheratoms.Thisresults indifferentintensitiesatI h,k,landI h,k,l.Asaresult, FPH+andFPH aredifferent.Withnoanomalousscattering,thelocationofFPH+ AndFPH (labeledingreen)areidenticalandwouldhaveidenticalintensities. Thesedifferencescanbeusedtoidentifytheanomalousscattererandgenerate phasesfortheproteinjustasinSIR/MIRmethods.

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