Informationaboutthepracticals

Practicalsdonebyeverybody
P1,P2SettingupNMRexperimentsandoptimizingparametersatthespectrometer
Thispracticalwillbedoneingroupsof45studentsatthespectrometer.Wewilldiscussandperform
calibrationofprotonandheteronuclearpulses,selectivepulses,waterflipback,radiationdamping,etc.
Basicbuildingsequencebuildingblockswillbesetupandanalyzed.

P3CCPN/Analysis(Mott,Warner)
InthispracticalwewilllearnthebasicsoftheCCPNanalysissoftwareanduseittosequentiallyassigna
smallprotein.Inthesecondpartofthepracticalwewilllookattheanalysisofrelaxationdata(T1,T2and
heteronuclearNOEexperiments)intheAnalysisprogram.

P4NMRPipe(Delaglio)
NMRPipe is a system for processing and analyzing multidimensional NMR spectra. Use of NMRPipe
requiresbasicknowledgeofUNIX/Linux,andtheabilitytocreateandeditshellscripts.Theprimarygoal
ofthepracticalsessionistoexplainandreproduceallthestepsneededforusingNMRPipetogofromraw
data on the spectrometer to a fully processed spectrum and peak table. Topics will include format
conversion and chemical shift calibration, phase correction, baseline correction, interactive spectral
graphics,peaktableediting,quantificationbylineshapefittingforindividualspectraandseries,andNon
UniformSampling.

P5ARIA(Malliavin,Nilges)
TheARIApracticalisdevotedtothepresentationanduseoftheARIAsoftwaresolutionforthecalculation
ofproteinNMRstructure.Inthispractical,wewillcalculatethestructureoftheTudordomain(Selenko
P,SprangersR,StierG,BuhlerD,FischerU,SattlerM.,2001,NatStructBiol8:2731)withARIA2.Thedata
comprise two NOESY specta, torsion angles from coupling constants, hydrogen bond restraints, and
residualdipolarcouplings.

P6CYANA(Gntert)
IntheCYANApractical,wewilldoautomatedresonanceassignment,automatedNOEassignment,and
structurecalculationforasmallprotein.AutomatedresonanceassignmentwiththeFLYAalgorithmuses
unassignedinputpeakliststodeterminethebackboneandsidechainchemicalshiftassignments.These
resonance assignments and unassigned NOESY peak lists are input for the subsequent automated
assignment of NOE distance restraints, which is performed iteratively in conjunction with structure
calculationsusingtorsionangledynamics.

P7RDCs&NMRrelaxation:practicalaspectsatthespectrometer(Grzesiek)
Wewillpresentdifferentalignmentmediaanddemonstratethealignmentbymeasuring2Hsplittingand
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DNHinselectedproteinsamples.

Wewillalsoshowasimplemeasurementof1HNT2timesbythe11spinechosequenceaswellsasbasic
experimentsformeasuring15NT2andT1inatwodimensional1H15Ncorrelationexperiment.

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P8RDCs&NMRrelaxationdataanalysis(Tjandra)
Wewillwalkyouthroughtheprocessofanalyzingarelaxationdatawithvariousdiffusionmodels.Wewill
alsodiscussdifferentaspectsofyoursamplethatwillinfluencetheanalysis,evaluatingwhichmodelis
thebesttodescribeyoursample,andgeneralguidelinesaboutrelaxationanalysis.

WewilldiscusshowtoestimateanalignmenttensorfromasetofRDCvalues.Wewillwalkthrougha
simpleprocessoffittingasetofRDCvaluestoastructure,evaluatingthegoodnessofthefit,anddiscuss
generalguidelinesinanalyzingRDCdata.

P9RDC&NMRrelaxation:MODULE&TENSORprograms(Salmon/Blackledge)
ThispracticalwillintroducethegraphicalsoftwarepackageModulefortheanalysisandexploitationof
residual dipolar couplings in terms of molecular structures. The practical is organized so that the
particuliaritiesoforientationalaveragingrelatedtoexperimentalparameterssuchasRDCs,thatdepend
onsecondranktensorsareintroducedgraduallytothestudentviarealisticexamples.Theuserdiscovers
theidiosyncrasiesoftheseexperimentalrestraintsasthepracticalgoeson.

Bothsimulatedandexperimentaldataareused,fromproteinsandnucleicacids.Aspectssuchastensor
determination,experimentalunderdetermination,angulardegeneracyandtranslationaltransparency,
andcombinationwithdistancedependentconstraintsaredealtwithandexperienceddirectlybytheuser,
overaseriesof6differentexercisestreatedhandsonbyeachofthestudents.

P10TALOS&more(Delaglio)
We begin the practical session with a talk covering two topics. The first topic is a discussion of NMR
homologysearchmethodsforextractingproteinstructureinformationfromchemicalshifts,andrelated
methodsusingdipolarcouplings.ThesecondtopicisuseofPrincipalComponentAnalysis(PCA)tohelp
characterize1Dand2Dspectralseries,suchasthoseusedinNMRligandscreening.Afterthetalk,wetry
handsonworkwithcorrespondingapplicationsincludingTALOSN(predictionofproteinbackboneand
sidechainanglesfromchemicalshifts),SPARTA+(predictionofchemicalshiftsfromproposedstructure),
CSROSETTA(abinitioproteinstructurefromchemicalshiftsandsequence)andspecView(interactivePCA
for1Dand2Dspectralseries).

P11Structurevalidation,CCPN(Vuister,Gutmanas)
ThevalidationpracticalwillbuildontheprecedinglectureandexploretheconceptsofNMRstructureand
data validation in more depth. The students will use the prerelease versionof the wwPDB validation
reports, and CING and PSVS validation portals to answer questions on the quality of three example
structuresfromthePDB.Thequestionswillfocusontheknowledgebasedgeometryvalidationofthe
structure itself, validation of the chemical shift assignments and analysis of conformational restraints,
whicharedepositedalongwiththestructure.Theaimofthepracticalistoemphasizetheimportanceof
validationoftheunderlyingdata,thequalityofthemodelandofthefitbetweenthemodelandthedata.

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Optionalpracticals
P12SPARKY(Jonker)
ThispracticalwillintroducethegraphicalsoftwarepackageSparkyforNMRspectraanalysis.Itwillstart
with the basics on the project setup, data conversion and the software handling (such as the menu,
shortcuts,spectraviewingandsynchronisation).Thecoursewillshowhowtooverlayspectra,howtodo
peakpicking,howtointegrateandassignpeaks,howtoworkwiththepeakandresonancelistsandhow
to work with 3D spectra. Additional features, which directly connect spectra to structure (chimera,
assignmentdistances)willbeshown,aswellastheanalysisofrelaxationdata(T1,T2)Severalexercises
willenabletheparticipanttoworkonNMRspectraandexplorethepossibilitiesofthesoftware.

P13StructurecalculationofRNA(Jonker)
Inthispractical,theparticipantswillcalculatetheNMRsolutionstructureofa14merhairpinRNAusing
the WeNMR ARIA webportal. The course will start with a general introduction about RNA structure
calculation (such as the nomenclature, RNA motifs, sugar pucker, backbone and base conformation,
restraintsandtheforcefield)followedbyanexerciseinwhichtheparticipantswillsubmitNMRdatato
thewebportal.TheresultsoftheRNAstructurecalculationwillbeanalysedandtheinfluenceofdifferent
datasetswillbeinvestigated.

P14NMRVIEW(Dames)
The NMRView practical provides in the first part an overview of the basic features of the software
(opening & displaying spectra, superimposing spectra, referencing of a 3D to fit areference 2D HSQC
spectrum,buildingofpeaklists,readingasequence,savingeverythingasstarfileetc.).Inthesecondpart
itisshownhowtoanalyze15Nrelaxationdata(T1,T2,NOE).ThethirdpartdealswiththeanalysisofNMR
titrationdataandthedeterminationofthedissociationconstantfromit.Inthefourthparttheuseof
NMRVIEWforthebackboneassignmentwithaspecifictoolisillustrated.Ofcoursetheprogramcanalso
beusedtoassignsidechainnuclei.

P15HADDOCK(Mott)
Inthispracticalwewilllookathowtousechemicalshiftmappingdatatogeneraterestraintsfordocking
complexesusingHADDOCK.WewillalsolookathowtoassessthequalityoftheresultsoftheHADDOCK
runandhowtochoosethebestclusterofmodels.

P16Structurecalculationswithunusualligands(Habazettl)
The aim of this practical is to introduce to structure calculation with ligands and NMR restraints. For
calculationsusingXPLORNIHthetopologyandparameterfilesfortheligandsneedtobegenerated.In
three different examples you learn how to proceed: First we start from the coordinates of a ligand
includingallhydrogens,whichissearchedanddownloadedfromthePDBdatabase.Inthesecondcase
theligandcoordinatesarefromaXRAYstructurewithoutprotons.Inthethirdexamplewecreateanew
ligandandcalculatethestructureoftheligandincomplexwiththeproteinbasedonNOEs,RDCs,and
dihedralangleswithasimulatedannealingprotocol.

P17SettingupHNCOBuildHNCOpulsesequencefromscratch(Grzesiek/Tjandra)
Wewillbuilda3DHNCObyastepbystepprocedure,startingfromsimpleprotondetection,followedby
introductionofthe1H15NoutandbackINEPTtransfer,the15N13CoutandbackINEPTtransfer,the13C
evolution,andthe15Nconstanttimeevolution.Yourproductoperatorsatwork!

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P18AdvancedNMRonlargeproteins(Riek)
Inthispractical,theparticipantswilllearnhowtomeasureandtransformNMRspectraoflargeproteins.
Inlargesystems,thewaterhandling,thetypeofmeasurements(i.e.TROSYandCRINEPT),thepolarization
transfers,therecoverytime,andtheevolutiontimeshavetobeoptimized.Inaddition,theselectionon
theFouriertransformationisimportantforoptimalsignaltonoise.Thispracticalwillbedoneingroups
of45studentsatthespectrometer.

P19PREs:experiments,dataanalysis,structurecalculations(Simon,Madl,Warner)
Inthispractical,wearegoingtocoverdifferentaspectsofobtainingstructuraldatafromparamagnetic
relaxationenhancements(PRE)byanalyzingdatafromaspinlabelcovalentlyattachedtoaproteinor
presentinsolution.WewilldiscussdifferentapproachestoperformingtheNMRexperimentsanddiscuss
theexperimentalsetup,includingcriticalexperimentalsettings.Inthefirstexample,wewillbrieflydiscuss
practicalaspectsofengineeringcysteineresiduesinto(oroutof)aproteinforspinlabelattachment.Then,
we will analyze 1H,15N HSQC spectra obtained with nitroxyl spinlabels covalently attached to the
engineeredcysteineresidues.FromtheNMRPREdata,wewillderivedistancesanddynamicinformation
forthespinlabelandshowhowtousethosedatainastructurecalculationprotocolwithinaria/cns.In
thesecondexample,wearegoingtoanalyze1HT1relaxationdataobtainedatseveralconcentrationsof
thesolubleparamagneticagentGd(DTPABMA)inordertoobtainsolventPRE(sPRE)data.ThesPREdata
willthenbeusedfordenovostructurepredictioninsPREROSETTA.

P20SolidstateNMRintroductionatthespectrometer(Sarkar,Reif)
Solid state NMR (ssNMR) deals with the same interatomic interactions such as chemical shift, dipolar
coupling,scalarcoupling,quadrupolarcoupling,whichareencounteredinsolutionNMRaswell.However,
duetotheabsenceofoveralltumblingmotion,theanisotropiccomponentsoftheseinteractionsare
presentinsolids.Asaresult,ssNMRmethodssignificantlydifferfromitssolutioncounterpart.Duringthis
practical,wewouldliketopointoutsomeofthesedistinctfeaturesofssNMR:(A)Importanceofsetting
upthemagicangle(with 79BrresonancesinKBr)accuratelywhilespinningthesample.(B)Polarization
transfer between heteronuclei using Hartmann Hahn cross polarization and the need for high power
decoupling.(C)Recouplingofhomonucleardipolarinteractionthroughtheresonanceofthesample
spinningfrequencyandthechemicalshiftdifference(D)Distancemeasurementbetweenheteronuclear
spinsusingrecouplingofthedipolarinteraction.

P21Relaxationdispersionexperiments:setupanddataanalysis(Asami,Petzold)
Inthispracticalsessionwestartwithashortintroductiontorelaxationdispersionexperiments,followed
byabriefdiscussionofthebestexperimentswithrespecttothedynamicquestionforbothproteinand
RNA.Wegivesomegeneralinformationaboutexperimentalsetupandthemostimportantdetails,which
needtobetakenintoaccount.Intheend,wewillfocusondataprocessingandanalysis,includingsome
tipsandtricks,whenthingsget"tricky".

P2213CdetectedNMRexperiments(Felli)
Recent improvements in instrumental sensitivity have brought 13C direct detection in the appropriate
sensitivity regime for applications to proteins. A whole set of multidimensional NMR experiments,
generallyreferredtoasexclusivelyheteronuclear,hasrecentlybeendeveloped.Theseexperimentscan
be used as a general tool in conjunction with 1H detected NMR experiments to study proteins; they
providecomplementary,andinsomecasesadditionalunique,information.

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The practical will focus on the specific aspects to consider in the experimental setup of 13C detected
experiments. The different experimental variants available (homonuclear decoupling method, starting
polarization source, longitudinal relaxation enhancement, etc.) will be presented considering as an
examplethebasic2Dand3DCdetectedexperiments.StudentswillhavethechancetoplaywithNMR
dataacquiredondifferentkindsofproteinsand,ifpossible,totrytheexperimentalsetupinfrontofthe
spectrometer.SinceCdetectedexperimentsresultveryusefulforthestudyofIntrinsicallyDisordered
Proteins(IDPs)nearphysiologicalconditions,aseriesofmultidimensionalexperimentsacquiredon
synucleinwillbeshownasanexample.
FelliI.C.,PiaiA.,PierattelliR.,AnnualReportsinNMRspectroscopy,2014,359418
FelliI.C.,PierattelliR.,J.Magn.Reson,2014,241,115125
BrutscherB.,FelliI.C,GilCaballeroS.,HoekT.,KmmerleR.,PiaiA.,PierattelliR.,SlyomZ.,IDPbyNMR,Springer,
inpress