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BiomolecularNMR

NMRstructure
determinationofRNA
HenryJonker

CenterforBiomolecularMagneticResonance(BMRZ)
InstituteforOrganicChemistryandChemicalBiology
GoetheUniversity FrankfurtamMain(Germany)

NMRsolutionstructurecalculationofa14merRNAusingCNS HenryJonker(2013)

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BiomolecularNMR

Introduction
RNAandDNAnomencluatureandstructuralmotifs
NMRrestraintsforRNAandDNA
Structurecalculation,simulatedannealing,forcefield

StructureCalculation
CNS(CrystallographyandNMRSystem)
Inputfiles,procedureandsetup
Structurecalculation

StructureAnalysis
Impactofthedataset
MOLMOLandPyMOL

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NMRforDNAandRNAstructuredetermination

BiomolecularstructuresdepositedinthePDBandNDB:
only5%DNAand3%RNA

VerystrongcontributionofNMR
18%oftheentriescontainingDNAand41%oftheentrieswithonlyDNA
23%oftheentriescontainingRNAand51%oftheentrieswithonlyRNA

Nucleicacidcomplexes
95%ofthelargecomplexesofnucleicacidswithproteinsaresolvedbyXray
crystallography,but40%ofthesmallercomplexes(<40kDA)byusingNMR

Commonlyusedsoftware
ARIA/CNS,CYANA,XPLORNIH,AMBER,HADDOCK

Statisticsfrom:Dominguezetal.,Prog.Nucl.Magn.Reson.Spectrosc.58,161(2011)
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DNA&RNAatomnomenclature
H5 C7H7#
1lettercodes:
O O
Hoogsteen H61 H62 5 H6 5 H6
Edge N 4 6 4 6
U A:Adenosine(ade)
T
N
3 1
N N
3 1
N C:Cytosine(cyt)
H3 2 2
H3
N
6
G:Guanine(gua)
7 N
H8
5
A
1
T:Thymine(thy)
8
9
4 2 O O U:Uracil(uri)
3
H2 Watson Sugar Watson Sugar
N N Crick Edge Crick Edge
N:Anybase
Sugar Edge
Adenine Uracil Thymine R:Purine(A,G)
WatsonCrickbasepair
Y:Pyrimidine(C,U,T)
H41
H5
N ...O M:A,C W:A,U,T
H42 H6
Hoogsteen 4
5
6
O P O S:G,C K:G,U,T
O O
Edge
3
C 1 5' A,U,T,C,G
N N
2 H5'1 - C - H5'2 B:notA H:notG
N
6
H1 4' O
7 5 1
N H3' H2'
1' D:notC V:notT,U
H8 8 G O
H4' 3' 2' H1'
4 2
9 3 H22 Sugar
N N N Watson Edge O3' O2'-HO2' (RNA)
Crick or H2'' (DNA: H2'1 & H2'2)
H21
Sugar Edge
Guanine Cytosine (Deoxy)Ribose
WatsonCrickbasepair

IUPACIUBJCBNandNCIUB,Eur.J.Biochem.131,915(1983),Eur.J.Biochem.150,15(1985)
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Secondarystructuralmotifs

singlestrand regularduplex mismatch singlebasebulge internalloop internalloop

(symmetric) (asymmetric)

bulgeloop hairpinloop 3wayjunction 4wayjunction looploopcontact pseudoknot

A(3)
5' U C
L3 A(2)
G=C

G(1)
L1 U G
A 3' L2 N
N U AG AG N M 'Uturn'
R A R
GA A
G A 3'
U AG Y G A
C
C 5'
C
5'
C
5' 3' PDB:1HC8 3' 5' PDB:1JJ2 5' 3' 5' 3' PDB:1KOC U 3' PDB:1KOG

Uturn Kturn Tetraloopmotifs Cloop

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Regularduplexconformations
31

34

45
28
23

26

20

18

Aform Aform Bform Zform

DNA RNA DNA DNA

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Basepairing H6
N
6 1
H5 5
U 2
O
AUreverse
Reversebasepairs 4 3 Hoogsteenbasepair
N H61 H62
relativeglycosidicbondisreversed N
O H3
Wobblebasepair
N
nonWatsonCrickbasepair 7 5
6
1
N
H8 8 A
Hoogsteenbasepairs 9
4
3
2
H2
N N
paringwiththehoogsteenedge
andmanymore....
H5
O N
O H6
5 H6 1
2 6
4 6
U 3
U 5
3 1
N N N 4
2 H5
H3 H3
O O
O O
N H1 N H1
6 6
7 5 1
N 7 5 1
N
H8 8 G GUwobble H8 8 G GUreverse
4 2 4 2
9 9
3 H22 3 H22
N N N
basepair N N N wobblebasepair
H21 H21

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Basetriples H6 C7H7#

6 5
N 1
T 4 O C7H7#
2 3 O
N H61 H62 5 H6
4 6
H3 N
O
3
T 1
N N
H3 2
N
6
7 5 1
N
H8 8 A O
parallel(YRY) 9
4
3
2

N H2
N

H6 H5 Examplesofbasetriples
PDB:1D3R
6 5 H41
antiparallel(YRR) N H41
1
C 4 N H5
2 3 H42 N
N H42 5 H6
4 6
O
O C
Extendedpurinestrandscanlead H+
N
3 1
N
2
toformationoftriplexDNA N
7
6
N
H1
5 1

Thethirdstrandcaneitherrunparallel H8 8 G O
4 2
9 3 H22
orantiparalleltothepurinestrand N N N

H21

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Specialstructuralmotifs H8 H21

N N
8
N
N N H22 3 9
2 4
9 7
G 8 H8
1 5 7
N 6
4 5 H1 N
H41
H5 N 3 G 6 O
N 2 1 O H22
H42 5 H6
N N H21
4 6
O H1
C H1
3 1 H21 N N
N N
2
H22 O 1 2
H+
2
N N O 6 G 3 N
1 3
C O N H1 5 4
6 4 6
7 5 1
N
5
H6 H42
N H8 8 G 7 9
4 2
9 3 H22 N N
H5 8
H41 N N N

H21 H8

PDB:1EL2 PDB:352D

imotif Gquadruplex

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Movementofbasepairs

movinginconcert rise stagger

tip,inclinationand(x/y)displacement
interbasepair slide stretch
rise,shift,slide
roll,twist,tilt
shift shear
intrabasepair
stagger,shear,stretch
propellertwist,opening,buckle

roll propellertwist
stagger buckle
rise z inclination tilt
3' 5'
tip roll slide
twist opening
y
propeller stretch
5' twist x 3'
shift twist
shear opening tilt buckle

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WhatisspecialaboutDNAandRNA?

Diversity
manydifferentstructuralmotivesandbasepairingpossibilities
hybridsandinteractionswithotherbiomoleculesandligands

Canadoptwelldefined3Dstructureswith(dynamical)biological function
riboswitch,ribozyme,ribosome,aptamerRNA

Catalytic(unnatural)RNAs
DielsAlderRibozyme
SELEX(SystematicEvolutionofLigandsbyEXponentialEnrichment)

Ribosome DielsAlderRibozyme L11RNAthiostrepton

Ramakrishnan(Cell,2002) Jschkeetal.(2005) Jonkeretal.(2007)

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DifficultiesforRNAstructurecalculations:
H2',H3'
H4',H5'/''
lowprotondensity&poordispersion
resonanceoverlapinsugarregion H2/6/8 H1',H5 CH3

RNA
highdegreeoffreedom
H3' H2'/''
6backbonetorsionangles
DNA
ribosesugarpucker
1H (ppm) 7 6 5 4 3 2 1
sugarbaseconnection

Bacbone,sugarand C2endo(Bform) C3endo(Aform) Glycosidictorsionangle

basetorsionangles (canonicalDNA) (canonicalRNA) Anti(~200),Syn(~60)

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15N&13CchemicalshiftsinRNA

13C(ppm)
1H,15NHQSC
C 5'

15N(ppm)
C 3'
amino &C2'
N4(C),N6(A)
&N2(G) C 4'
imino
N3(U)&N1(G)

C 1' 1H,13CHQSC

1H(ppm) 1H(ppm)

N7 N1 N3 N9 N3 N1 N4 N6 N2
(A,G) (A) (A) (A,G) (U) (C,G,U) (C) (A) (G)

15N(ppm) 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70

C4 C2 C6 C2 C4 C6 C8 C5 C5 C5 C1' C4' C2',C3' C5'

(C,U) (C,U) (A,G) (A,G) (G,A) (C,U) (A,G) (A,G) (U) (C)

13C(ppm) 170 160 150 140 130 120 110 100 90 80 70 60 50

Spectrafrom:Frtigetal.,Chembiochem4,936962(2003)
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RNAtorsionanglesseparatedbysugarpuckarconformation
1000 1000 1000

C3'- endo 500 500

500 0 0
60 60
C2'- endo
30 30
0 0 0
0 60 120 180 240 300 360 0 60 120 180 240 300 360 0 60 120 180 240 300 360
1000 1000
pseudorotationphaseangle(P)and
maximumpuckeringamplitude(Vmax) 500 500

(V4+V1) (V3+V0) 0 0
tan(P)= 60 60
2V2 (sin36+sin72)
30 30

Vmax =V2 /cos(P) 0 0

0 60 120 180 240 300 360 0 60 120 180 240 300 360
1000 1000

500 500
C3' endo
0 0
DNA+RNA RNA 60 60

30 30
C2' endo
0 0
0 60 120 180 240 300 360 0 60 120 180 240 300 360

Thehistogramsshowtheoccurrenceofthespecifictorsionanglesin23SrRNA
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ExperimentalNMRrestraintstodeterminetheRNAstructure

Resonanceassignment
preferablycompletewithassignmentof2'OH
Distances,basepairsandhydrogenbonds
NuclearOverhauserEffect(NOE)from2D/3DNOESYspectra
hydrogenbondsfromH2J(HN,N)fromHNNCOSY
TorsionAngles
backbone(,,,,),base()andsugar(12&34)angles
fromJcouplingsandcrosscorrelatedrelaxation(CCR)rates
ResidualDipolarCouplings(RDCs)
pf1phagealignment,fieldalignment
sugar(H1'),base(H6,H8)andimino(H1,H3)

ForceFieldconstraints
Nucleicacidtopologyandparameters
atoms,bonddistances,connections,angles,stereochemistry,charges,vanderWaalsand
electrostaticinteractions,harmonicpotentialenergyfunctions, Coulomb,LennardJones,...

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NMRstructurecalculations

Energypotentials(fortheforcefieldparametersandNMRdata)
Structurecalculationprogramstryto"fold"amoleculeintoa3Dstructurethatagrees
withtheexperimentalNMRrestraints andtheforcefieldconstraints
Minimizationofthedifferences,manifestedasenergyviolations,drivethemolecule
towardsitsconformation
Thetargetenergylandscape isthesumofsquares(orsimilar)oftheviolationsandhas
manylocalminima

Targetenergyfunction

Etotal =Ebonds +Eangles +Edistances +Erdcs +E...

E :energyfromforcefield/restraintviolation

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MolecularDynamics(MD)

Directionofmotiondependsonforces(restraintsandforcefield)andmomentum
CartesianAngleDynamics (CAD,coordinatespace)
(timestepforflexiblebondsandvibrations:25fs)

Energy
TorsionAngleDynamics (TAD,angularspace)
(about10timesfaster lessdegreeoffreedom)
MDcanovercomelocalenergybarriers
temperaturerelatestokineticenergyandthusvelocity
scalinganduseofdifferentenergyterms
(increaseofrestraintenergytermsduringcooldown)
X
Use SimulatedAnnealing (SA)protocol
Temperature

initialMD refinement
coolingdown refinement
10.000steps 8.000steps
at10.000K at2.000K 5.000
stages (inexplicitsolvent)
stepsto
1.000K 10.000stepsto50K Time

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The14merRNA:

Thermodynamicalstablehairpinmotif 7
meltingtemperatureof74C U7 C8 U C8
6
verystablecUUCGg
tetraloop(YNMG U6 G9 U G9
motifwithaCG
5
closingbasepair) C5 C G10
G10
4
A U11
transwobbleU6G9
3
G12
O 2 N1
basepairincluding U6 C
O2'hydrogenbond
O6
G9 2
O2'
G C13
Easycommerciallyavailable
1
ao.SilantesGmbH(isotopelabeled) G C14
VerysuitableasmodelsystemforNMR:
structuralanddynamicalinvestigationsandmethoddevelopment
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NMRrestraintsforthecalculation

NOEdistances 14mer_noe.tbl
DihedralAngles 14mer_dihedral.tbl
RDCs 14mer_rdc.tbl
Hydrogenbonds 14mer_hbond.tbl
Baseplanarity 14mer_baseplan.tbl

Forcefieldconstraints(includingprotons)

topology dnarnaallatom.top
parameters dnarnaallatom.param
linkage dnarnapho.link (includes5'phosphategroup)

tensorparameters axis.param (forusewithRDCs)

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RNAstructurecalculationexercise

Calculatethestructureofthe14merRNA.
Impactoftorison
anglesandRDCs
Group1: NOEs+basepairs
Group2: NOEs+basepairs+RDCs
Group3: NOEs+basepairs+Dihedrals
Group4: NOEs+basepairs+Dihedrals+RDCs

Seetheimpactofdifferentdatasets

Whatisthe RMSD of10structureswiththelowestenergy?

ComparetheNMRsolutionstructurewith Xray
Whatarethedifferences?
Isthetranswobble basepairpresentinthesolutionstructure?
Measurethehydrogenbond lengthoftheU6G9&C5G10basepairs

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Directorysetup availablefilesinthedatadirectory

Restraintfilesandinputdata
NMRdata(.tblrestraintfiles)
Sequence:14mer.seq
data
data
Tensorfiles:ani.pdb/ani.mtf
XraystructureofthecUUCGgloop:1F7Y.pdb

CNSscriptstoprepareinputfilesandrunthecalculation
generate_seq.inp
run1
run1
generate_extended.inp
merge_structures.inp
anneal_cv.inp

run# run2
run2
Createarundirectory foreachcalculation(eg.run1,run2,...)

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StructurecalculationwithCNS (CrystallographyandNMRSystem)

Online userinterface: http://cnsonline.org/v1.21/

Usetheprovidedinputfilesfromthedatafolder!

upload an existing
input file

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Procedure

Sequence(.seq) Theonlyinformationatthebeginning
GGCACUUCGGUGCC
generate_seq.inp

Topologyfile(.mtf) Descriptionofallatoms,bondconnectivity,
chargesandangles
generate_extended.inp

Extendedstructure(.pdb) Extendedmolecule

merge_structures.inp
Z

Extendedstructure+tensor AddalignmenttensorforRDCs OO Y
X

anneal_cv.inp

Runcalculation RestrainedTAD

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Generatetopologyfile

Editgenerate_seq.inp

n.a.sequencefile
14mer.seq
outputstructurefile
14mer.mtf
Checkhydrogenflag
true

Checknucleicacid
forcefield
dnarnaallatom.top/paramanddnarnapho.link (withrelativelinkCNS_TOPPAR:)
Saveandruntheprotocolscriptinthedatafolder
cns<generate_seq.inp>generate_seq.out
moleculartoplogyfilewillbegenerated

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Generateextendedstructure

Editgenerate_extended.inp

structurefile
14mer.mtf
forcefieldparameterfile
dnarnaallatom.param
outputcoordinatefile
14mer_extended.pdb

Saveandruntheprotocol
scriptinthedatafolder
cns<generate_extended.inp>generate_extended.out
pdbcoordinatefilewillbegenerated
HavealookattheextendedstructureusingMOLMOLorPyMOL

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Addalignmenttensor(RDCs)

Editmerge_structures.inp

structure&coordinatefiles
14mer.mtf
14mer_extended.pdb
ani.mtf,ani.pdb
segidforthetensor:ANI
tensorincrement499
toendupwith500
outputfiles
14mer_ani.mtf
14mer_ani.pdb
Saveandruntheprotocolscriptinthedatafolder
cns<merge_structures.inp>merge_structures.out
moleculartopologyandpdbcoordinatefileswiththerna&tensorwillbegenerated
Havealookattheoutputfiles

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SetuptheRNAstructurecalculation

Createanewrundirectoryandcopytheanneal_cv.inpfile
cd.. (goonedirectorybelowdata)
mkdirrun1
cdrun1
cp../data/anneal_cv.inp.

Openandedittheannealingprotocol:anneal_cv.inp
checktheinputfiles
thenameoftheinputfilesshouldincludethecomplete(relative)pathtothedatafolder!
eg."../data/filename"

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Edittheannealingprotocol (structure/atomsselection)

Forcefieldparameterfiles:
dnarnaallatom.param
axis.param

Moleculartopologyfile:
14mer.mtfor
14mer_ani.mtf(RDCs)

Coordinatefile:
14mer_extended.pdbor
14mer_ani.pdb(RDCs)

Changethebackboneatomselectioncriteriatonucleicacids

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Edittheannealingprotocol (refinementparameters)

MolecularDynamics:
torsionforhotphase
torsionforcoolphase

Randomseesnumber:
Takeyourluckynumer:)

Numberofstructures:
accepted
50

Donotcalculatean
averagestructure

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Edittheannealingprotocol (annealingsettings)

UsethestandardSettings
assuggestedforDNA/RNA
nochangesneeded
forthispractical

Usea2ndslowcoolingstage
withcartesiandynamics
usethestandardvalues

Finalminimizationstage
usethevaluesasgiven

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Edittheannealingprotocol (crossvalidation,NOEdata&hydrogenbonds)

CompleteCrossValidation
false

NOEdata
14mer_noe.tbl

Hydrogenbonds
14mer_hbond.tbl

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Edittheannealingprotocol (RDCs,otherdataandoutput)

SusceptibilityAnisotropy
RDCdata(onlygroups2,4)
14mer_rdc.tbl
axial:8.62
rhombicity:0.13

Dihedralanglerestraints
14mer_dihedral.tbl
(onlygroups3,4)

Baseplanarityrestraints
14mer_baseplan.tbl

Baseoutputname
14mer

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RuntheRNAstructurecalculation

Carefullychecktheanneal_cv.inpfileandinputdata
correctnamesandpaths?

Saveandruntheprotocolscriptintherun#folder
cns<anneal_cv.inp>anneal_cv.out
structurecalculationwillstartandpdbcoordinatefileswillbegenerated
calculationmessagesarearchivedintheanneal_cv.outfile(checkforproblemsanderrors!)
mostlyerrorsoccurduetowrongfilenamesandtypos(missingletters,spaces,etc)...

Selectthe10lowestenergyacceptedstructures
readouttheoverallenergyofeachstructureandcreateasortedlist(overall.txt)file
grepioverall14mera*.pdb|sortrg+3>overall.txt
takethe10structureswiththelowestenergyforfurtheranalysis

33

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

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

Analysis

CalculatetheRMSD(rootmeansquaredeviation)value
openthe10lowestenergystructures
selectbackbone(bb)
selectresidues:213
edit,fit,tofirst,ok
RMSDvalueappearsatthebottom
CalculatetheRMSD(bb/heavy)for:
residues:213,theloop:510
thestem:25,1013

OpentheXraystructure1F7Y.pdb
theresiduenumbersinthisPDBfilehave
beenadaptedinadvancetofitwiththe
14mersequence
fitthestructures
selectbb,residues510,edit,fit,tofirst
Comparetheloopstructures

34
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

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

Analysis

OpenastructurewithPyMOL
Checkbasepairs&planarity
Hydrogenbond distance
wizard,measurement,distance
select1stand2ndatom
TransWobble U6G9
U6 (O2') G9(O6)
U6(O2) G9(N1)
WatsonCrickC5G10
C5(N4) G10(O6)
C5(N3) G10(N1)
C5(O2) G10(N2)

35

BMRZ
FrankfurtamMain

www.bmrz.unifrankfurt.de
Goethe University

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

Structureofthe14merhairpinRNA
CalculatedwithARIA/CNS
Withdifferentsubsets(NOEs,RDCs&Dihedrals)
Refinementinexplicitwater(includedOPLS)

onlyNOEs NOEs+RDCs NOEs NOEs+RDCs

+dihedrals +dihedrals
overallrmsd:0.85 overallrmsd:0.69 overallrmsd:0.41 overallrmsd:0.37
(loop:0.45,stem:0.78) (loop:0.33,stem:0.71) (loop:0.27,stem:0.31) (loop:0.25,stem:0.30)

36
 BMRZ

FrankfurtamMain
www.bmrz.unifrankfurt.de

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

ImpactofNMRrestraints

Summary

1)NOEs 2)NOEs 3)NOEs 4)NOEs

+basepairs +basepairs +basepairs + basepairs
+RDCs +Dihedrals +Dihedrals
+RDCs

RMSD(bb)
residues 213

RMSD(bb)
loop510
RMSD(bb)
stem25,1013

37