BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

SPARKY
GraphicalNMRassignmentandintegrationprogram
forproteins,nucleicacids,andotherpolymers

HenryJonker

CenterforBiomolecularMagneticResonance(BMRZ)
InstituteforOrganicChemistryandChemicalBiology
GoetheUniversity FrankfurtamMain(Germany)

NMRdataanalysiswithSparky HenryJonker(2013)

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Sparky

Developed&maintainedbyDonaldKnellerandTomGoddard
(UniversityofCalifornia,SanFrancisco)
www.cgl.ucsf.edu/home/sparky/ (download&onlinemanual)
tech.groups.yahoo.com/group/nmr_sparky/ (discussiongroup)

Workssimultaneouslywithanynumberof2D,3Dor4Dspectra

Freelyavailableforallcommonoperatingsystems
MicrosoftWindows,Linux,SGIUNIX(IRIX)andMacintosh

CanuseallcommonlyprocessedNMRspectrafrom
XWinNMR,Topspin(Bruker),VNMR(Varian),NMRPipe,Felix
Easytoconvertandtoeditthepropertiesofthespectra

2
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Directorysetup:

Essentialfoldersandfiles(ineg.D:/SPARKYDATA/SUBSTANCE)

data
processedNMRspectrainsparky(.ucsf)format(remainsunmodified)
save
spectra(.save)fileswithallthedisplaysettings,peaks,assignments,labels,...
lists
outputforpeakandassignmentlists,spectra(postscript),...
projects
sessionproject(.proj)fileswiththerelationandsettingsofmultiplespectra

sparky.bat(windows)
notessential,butusefullexecutabletothestartsparkyproject
(copyfromsparky/binfolderandaddthevariable:SPARKYHOME=.)
checkthepythonversionfordifferentversionsofsparky

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Dataconversion:

Convertspectrato.ucsfdata:
example:bruk2ucsf 2rrn15hsqc.ucsf
willcreatean15hsqc.ucsffilefromthexwinnmr/topspin2rrfile
also:vnmr2ucsf forVarianspectraandpipe2ucsf forNMRPipespectra

Showspectruminformation:
example:ucsfdata hnca.ucsf
typeonly'ucsfdata'togetafulllistofalltheoptions

Editspectrumsettings(eg.axisnames,frequency,offset,....)
example:ucsfdataa2HNn15hsqc.ucsf
willrenamethew2axisto'HN')
example:ucsfdatap2on15noesy2dhh.ucsfn15noesy.ucsf
makesa2D1H1HNOESYprojectionfromthe3D1H1H15NNOESYHSQCdataset

4
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Menu&Pointer

Commandsareavailableviathemenuand2lettershortcuts.
rightclickinspectrumfordirectmenuaccess

Themousepointer
selectfunctionordirectlychangewithkeysF1F12

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Viewmode

Useviewoptions(vt)andcontourlevels(ct)tochangethelookofthe
spectrum.Peaksandthelabelsizescanbeadapted(oz).

crosshair
(also connectedto
other spectraviews)

contourscale
slicepanels(include integration)
axisscales(morefor3D,4D)
peakinfopanel

6
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Viewmode

Adapttheaspectratiotowidenor
narrowthespectrumview

Adapttheasssignmentformattochangethepeaklabels:
%a1%a2 fullatomnamesandgroupresidues G16NHN
%A1%A2 forcethegroupstobedisplayed(noesy) G16NG16HN
%G1 showonlyresiduename(offirstaxis) G16

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Move,Zoom,Duplicate,Hide&Center

Mousepointermodeorshortcutstozoomandcenter
zoomin(zi),zoomout(zo)
showfullspectrum(zf),zoomtopreviousstate(zp)
centerview(vc)onpeakposition

Additionalviewsofthesamespectrum
createaduplicateview(vd)
usefultomakestripsorviewa3Dspectrumfrommultiplesides.
takecarenottoremovetheoriginalviewinsteadoftheduplicate!
(abadsituationmayarriseinwhichcommandsmaynotwork)

Managespectrumviews
hideaspectrumview(vh)
showthespectrumbyselectingintheviewlist(pv)orinthewindowmenu.

8
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

OpenSpectra(exercise)

Convertabrukerprocessed2D1H15NHSQCdatasettotheSparkyformat
andchangethenameoftheamideaxis:
openterminaland:cd*sparkydatadirectory*/EMBO/bruker/4/pdata/1
bruk2ucsf2rrn15hsqc.ucsf
ucsfdataa2HNn15hsqc.ucsf

RunSparkyandopentheNMRspectrum(fo)
Changethecontourlevels(ct)to8e+6(lowest),10(levels)
Zoomout(zo),selectthe'find/addpeak'pointermode(F8)anddragaboxaroundall
thepeaksinthespectrum.

Howmanypeaksaredetected?(seeinmainsparkywindow)
Areallpeakspositionedwell?

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Synchronization

Synchronize(yt)theaxesof2ormoreselectedspectra
Theviewsoftheconnected2Dand/or3Dspectra
willmovesimultaneously

Alignspectra(al)witheachotherbymatchingpeakpositions
referenceonespectrumtoanother
thenamesofthesynchronizedaxishavetobethesame(usetheucsfdata)

10
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

OverlaySpectra

Overlay(ol)thecontoursofonespectrumontoanotherspectrumview
spectrumtypehastobethesame(dimension&axisnames)
thecontourlevelsofeachspectrumareseperatelyadjusted
overlayasmanyspectraontooneview
onlythepeaklabelsof
theoriginalviewareshown

11

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

PeakPicking

Peakscanbepickedusingthe'find/addpeak'pointermode.
manualorautomaticpeakpickingbydraggingaboxinaregionofyourspectrum.
(peakpickparametersettings(kt)canbeadjusted)
restrictedpeakpickingofa3Dspectrumfroma2Dspectrum(kr).
foldedpeakscanbealiasedtothecorrectfrequency.

Select,adjust,integrateandcopypeaks
Selectallpeaksinthespectrum(pa),centerthepeakpositions (pc).
Integrate(pi)thepeakvolume(s).Thesettingscanbeadjusted(it)andoverlapping
peakscanbefittedsimultaneously.
Copyandpastepeaksandassignmentsfromoneviewtoanotherusingornamentcopy
andpaste(oc,op).
Movepeakswithasameresonanceassignmentsimultaneous(mv).

12
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

PeakAssignment

Assigntheselectedpeakusingtheassignmentdialog(at)
Resonancesknowninthecurrentprojectandclosestinfrequency tothepeakposition
fortheselectedaxiswillbehighlightedandcanbeselected.Fornewresonancesthe
residuegroupandatomnamehavetobeenteredmanually.
Rename,mergeorswapresonances(rn).
Removeunusedprojectresonances(dr).

Assignmentguessing
Assistpeakassignment
basedonknownresonances
(selectppmrangesandthe
optionalgroupandatoms)
Thevaluesarealsousedby
theassignmentguessing
pointermode(shF2).

13

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Listsofpeaksandresonances

Everyspectruminsparkyhasapeaklist(lt)associated.
Doubleclickingonapeakinthislistwillshowandcenterthis peakinthespectrum(or
selectitandtypevc).

Eachprojecthasaresonancelist(rl)associated.
Theoutputiscustomisableandexportabletootherapplications.
Assignmenttable(tb)

14
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

OverlayandAssign(exercise)

Openthe'Tseries.proj'projectandsynchronizetheviews(yt)
Synchronizetheprotonaswellasthenitrogenaxisofallthe4 1H15NHSQCspectra
(recordedatdifferenttemperature).

Openthe'Overlayviews'dialog(ol)
Overlaythe298K,308Kand318Kspectraontopoftheoneat288K.

SelecttheHSQCat288Kandopenthepeaklist(lt)
Select(doubleklick)thepeaknamed'A6'.
Assign(at)thecorrespondingpeakfor'A6'inthe318Kspectrum.
Assignsomemorepeaksat318K(tryalsointhemorecrowdedregionofthespectrum
andcheckthepeaks'K3'and'G88')
Whathappensto'K3'and'G88'andhowwouldyouexplainthat?

15

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

3DSpectra

Theviewwindowcancontain3Dand4Ddata,butonlya2Dcontourplot
isshown.

Transpose(xx)androlltheaxis(xr)
13C
Swap(alsofor2D)orcycletheaxis.

Orthogonalviews(ov)
Directlygeneratesthreeorthogonal
(duplicate!)viewsofa3Dspectrum.

Viewdepth (vz)
Controlshowmanyplanesapeakcan
beawayfromthecurrentlydisplayed 1HN
planeandstillbeshown. 15N

16
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Stripplot

Showstripsfrom3Dspectrasidebyside(sp)

Stripsdialog(ss)
Selectthespectratobeshown.

Searchandmatchstrips(sT,sm)
Lookforotherstripsthatcontainpeaks
withmatchingresonances.

Backboneandsidechain
resonanceassignments

17

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

3DSpectra&Assignment(exercise)

Open'3Dhnco.proj'projectandsynchronizetheviews(yt)
SynchronizetheHN,15Nand13Caxisofallspectra.

Selectthe1H15NTROSYandopenthepeaklist(lt)
Select(doubleklick)'K3'tocenterthispeakinallspectraandassigntheCOresonance
ofthepreceedingresidue'A2'inthe2DHNCOusingthe3DHNCO. Dothesameforthe
next10residues.

CanoneassignV5HNK4COandQ8HNA7CO?
Ifnot,howwouldyousuggesttoresolvethis?

Makeastripplotofthefirst10residues(sp)
Selectthe3DHNCOspectrum(ss)andaddthestrips(sn).

18
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Backup&Print

Automatedbackups
Setthebackuptimeperiodinthe
preferencesdialog(pf)
The.savefileiscopiedto.save.BAK
The.projfileiscopiedto.proj.BAK
Onlyonebackupiskept.

Printaspectrum(lt)
Selectthespectrumandsave
asapostsciptfile
Printthisfile(useao.ghostview)

19

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

Chimera

DirectinteractionbetweenstructureandNMRdata(km)
Showpeakassignmentsand/orNOEsaslinesonthestructure.
Selectapeakinthespectrumandthecorrespondinglineinthemoleculeviewwill
highlight.Viceversa,selectalinetojumptothecorrespondingpeak.
Nomenclaturehastomatchandpseudoatoms(eg.Q/#)mayhavetobeadded
OnlyworksforcertiancombinationsofSparkyandChimera(eg.3.113with1.2143)

20
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

AssignmentDistances

AssistmanualNOEassignmentusingdistances(ad)
Listtheassignmentpossibilitiesforaselectedpeakbasedonchemicalshiftagreement
(ranges)anddistancescalculatedfromavailablestructure(s)or model(s).
Detectcloseatomswithoutpeakassignment(completeness)
Nomenclaturehastomatchandpseudoatoms(eg.Q/#)mayhavetobeadded

21

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

RelaxationData(eg.T1,T2)

Fittheexponentialdecayfromaseriesof2Dspectra(rh)
Entertherelaxationtimeparametersforeachspectrum(setup)
Selectoneormorepeaksandtype'rh'.
A"FitPlot"willpopupwhenselectingarow.
Selectadatapointinthe"FitPlot"
toshowthepeakinthecorresponding
spectrum.

22
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

Goethe University
Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

RelaxationDataAnalysis(exercise)

Open'RelaxationT2.proj'project
Open'Relaxationpeakheights'dialog(rh)
Selectallthespectrain'Setup'andentertherelaxationtimeparameters(in
milliseconds).Use25trailsfortheerrorestimation.Confirmwith'apply'.
Selectallthepeaksinthefirstspectrum(pa)
Type'rh'againandcheckthedataappearingintherelaxationdialog.Selectingaline
willshowthecurveandselectingadotonthecurvewillshowthecorrespondingpeak
andspectrum.

Whichregionofthisproteinismoreflexible?
Savetherelaxationlistforexporttootherprograms(eg.excel)
MakeaplotofthesequenceagainsttheT2relaxationtime

23

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

Network ofResearchInfrastructures
www.wenmr.eu providingAccess and Technological
www.bionmr.net Advancements inBiomolecularNMR HenryJonker
BiomolecularNMR

SpinGraph

Diagramofconnectedatomsforeachassignment(sg)
TheatomnamesmustberecognizedbySparky(definitionsareavailableforproteins,
RNAandDNA).
Directinteractionbetweenthespingraphandspectrumpeaks
Onecanmakeowntemplatesand/ordefinetranslations.
Differentcolorforeachspectrum.Customizablelayout.
SpinGraphAssignertool(ga)available

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