the varkud satellite 
 ( vs ) ribozyme is a catalytic rna which performs 
 cleavage and ligation of a phosphodiester bond , and it requires mg for tertiary structure formation and catalysis . though there is no available structure of the complete ribozyme , 
 several of the four hairpin loops and internal stems and junctions 
 have been characterized by nmr . stem - loop v ( slv ) undergoes a mg - dependent 
 conformational change which allows it to orient the substrate stem - loop 
 i ( sli ) via watson  crick base pairing in a kissing - loop interaction . the structure and dynamics of slv were examined by nmr revealing 
 a mg - dependent conformational switch of the loop conformation 
 ( shown in figure 1a , b ) . the mg - dependence is particularly interesting since 
 it illustrates a role for divalent ions in the association of sli 
 with slv . slv is a six base pair stem capped by a 
 five - membered loop with the sequence 5-u696g697a698c699u700 - 3 
 and it forms a canonical u - turn motif in the presence of mg . the structural features of a u - turn motif include a unr sequence 
 ( where n is any nucleotide and r is a purine ) ; a y : a , y : y , or g : a 
 loop - closing base pair ( y is pyrimidine ) ; a sharp turn of the backbone 
  torsion angle 3 to the u ; a hydrogen bond from u 2 
 oh to r n7 ( figure 1c , d ) ; a hydrogen bond from u nh3 to the phosphate oxygen 3 
 to r ( figure 1e , f ) ; 
 and stacking of the u with the r 5 phosphate group ( figure 1e , f ) . these canonical 
 features are illustrated in molecular graphics images of the experimental 
 structures in the presence and absence of mg in figure 1 panels c , d , e , 
 and f , respectively . in a monovalent ion environment ( -mg or mgfree ) , slv is described as a noncanonical or  loose  
 u - turn motif , and these characteristic intermolecular interactions 
 range from weak to nonexistent . in a divalent 
 ion environment these interactions are able to form and stabilize 
 a more compact loop . it has been proposed 
 that mg stabilizes the u - turn of slv and neutralizes 
 the electronegative charges in the loop , making it easier for the 
 substrate sli to bind . ( a ) the mgfree 
 most representative structure determined by nmr ( 1tbk ) . bases of the five - membered 
 u - turn are labeled and shown in color . ( b ) the mgbound most representative 
 structure determined by nmr ( 1yn2 ) . bases of the five - membered u - turn are shown in the 
 same color as part a , and hexahydrated mn are shown as 
 blue spheres . the primary sequence is shown with the bases of the 
 u - turn in black . close ups of the characteristic u - turn hydrogen bond 
 between the 2 oh of u696 ( yellow ) and the n7 of 
 a698 ( red ) ( c ) in the absence of mg and ( d ) 
 in the presence of mg . close ups of the characteristic 
 u - turn hydrogen bond between the 3 phosphate of a698 ( shown in purple here as the 5 phosphate of c699 ) and the n3 of u696 , and stacking of 5phosphate 
 of a698 with the u696 base in the ( e ) absence 
 and ( f ) presence of mg . solid lines indicate a favorable 
 interaction ( stacking or hydrogen bonding ) is present and dashed lines 
 indicate absence of interaction . the experimental evidence outside of nmr includes kinetic 
 and thermodynamic 
 characterization of sli and slv . a focus on the solvent exposed u700 base showed that mutation to another base maintained the 
 enzymatic rate of the wild type u , but deleting the base reduces kcat / km . similarly , mutating u700 to c showed 
 no change in binding affinity for sli , but deletion of u700 reduced the binding affinity . this led 
 to the hypothesis that u700 contributes to the enthalpic 
 stability of slv by donating a phosphate group that coordinates a 
 mg ion , and that the solvent exposed nature of the base 
 would contribute to increased entropy in the monovalent ( unbound ) 
 environment . slv is a rich source of information about mg - dependent 
 structural changes in small rna molecules , and so is of particular 
 interest for molecular dynamics ( md ) simulations . nucleic acid force 
 fields lag behind those for proteins , in part due to the high charge 
 density and high flexibility of the nucleic acid backbone . in rna  which adopts many types of structures 
 beyond a simple duplex  capturing the dynamic aspects , coupled 
 with the long time scales over which these conformational changes 
 can occur , is particularly challenging . however , recent modifications , 
 some specific to rna , have brought force fields into better alignment 
 with experimental data . here we use md simulations 
 to provide atomic - level detail into 
 the dynamics of ion association with slv . we show computational evidence 
 supporting the current hypotheses for the role of mg ions 
 in stabilizing slv , as well as the role of the u700 base . 
 additionally , we show that the dynamics of the unbound slv ( in a monovalent 
 ion environment ) adopt u - turn characteristics similar to the canonical 
 u - turn characteristics of the mg - bound conformation , 
 though admittedly not identical to the nmr structure . we propose the 
 slv rna in the presence of monovalent ions samples a dynamic and wide 
 range of structures , including a  gatekeeper structure 
 which is a precursor to mg binding , adopting structural 
 features which are more similar to the bound loop than to the excess 
 of unbound conformations . a flowchart 
 of the simulation protocol is provided in the supporting information as supporting figure 1 . were 
 taken from the first model of the nmr ensembles of slv : 1tbk ( mgfree ) and 1yn2 ( mgbound ) . the rna , without the ions that were present 
 in the pdb file , was placed with a buffer of 12  of water , resulting 
 in the addition of 5800 water molecules . for the mgbound systems eight mg ions were added back to neutralize the system 
 charge , and five additional mgcl2 molecules were added 
 to generate an 40 mm excess mg concentration . 
 for the mgfree systems 16 na ions were added to neutralize 
 the system charge and six additional nacl molecules were added to 
 generate an 50 mm excess na concentration . all 
 ion initial positions were randomized with placement at least 6  
 from the rna and at least 4  from each other by swapping positions 
 with random water molecules . joung simulations were carried 
 out using the amber12 and developmental 
 versions of the amber14 suite of programs using the gpu ( cuda ) 
 version of pmemd with spfp ( a mixed single / fixed precision model on 
 the gpu ) . the ff12 force field for nucleic 
 acid simulations was used , combining ff99 + parmbsc0 modifications 
 + chiol3 modifications for rna . periodic boundary conditions were used in all simulations , and the 
 particle mesh ewald method , with default parameters , was used to handle 
 electrostatic interactions with a 9  cutoff for direct interactions . as presented in the pdb , the original 
 nmr structures of slv  
 mg were described by an ensemble of 10 structures consistent 
 with the nmr constraints . our initial simulations used model 1 of 
 each ensemble as a starting structure , and to evaluate experimental 
 variability , 10 simulations were produced from that model 1 , with 
 different initial velocities ( via randomized random number generator 
 seeds using the ig = 1 option in amber ) as well as different 
 randomized initial ion positions . minimization 
 and equilibration procedures were the same as previously used . in brief , the initial minimization of coordinates 
 and heating to 300 k were followed by successive rounds of minimization 
 and equilibration performed with decreasing positional restraints 
 ( from 5 kcal mol  ) on 
 the rna . the system was determined to be equilibrated when the potential 
 energy , density , and pressure of the system stabilized . unrestrained 
 production md simulation runs of 250 ns were begun 
 from these equilibrated systems , and the properties of the rna and 
 ions were measured from this portion of the trajectory . production 
 runs were constant volume and temperature ; temperature was set to 
 300 k and regulated using the langevin algorithm with a collision 
 frequency of 5 ps . bonds to hydrogen were constrained using the shake algorithm , allowing 
 use of a 2 fs time step . the instability 
 of the slv rna in 50 mm nacl simulations described 
 above led us to rerefine the 1tbk nmr structure in explicit solvent using the published 
 nmr restraint data . rerefinement simulations were carried out in 50 
 mm nacl , with equilibration as described above for models 2 through 
 11 of the original ensemble ( 10 structures total ) . in addition to 
 positional restraints , nmr experimental noe distance restraints were 
 applied to the rna . reported nmr restraints in dyana format were translated 
 into amber format using the makedist_rst program , totaling 553 restraints ; 
 527 distance restraints with a force constant of 20 kcal mol  and 26 torsion angle restraints with 
 a force constant of 500 kcal mol rad were used throughout equilibration and rerefinement unless otherwise 
 noted . the position 
 of the center - of - mass of the molecule was reset to zero every 10 ps . to analyze the ensemble of 10 slv structures in the restrained 
 rerefined simulations , the cpptraj analysis program from ambertools 
 was used to cluster the structures with a hierarchical agglomerative 
 algorithm on all heavy atoms using an epsilon value of 1.0  
 with a sieve of 5 frames ( supporting script 1 ) . the representative structure from the largest 
 cluster in each of the 10 trajectories was minimized using 1000 steps 
 of steepest descent followed by 1000 steps of conjugate gradient , 
 with restraints intact to allow structures to relax with respect to 
 the nmr restraints . each minimized structure was subsequently subjected 
 to heating and annealing over 4 ns , followed by 6 ns restrained npt 
 dynamics ( supporting script 2 ) . during 
 this step coupling to the heat bath was lowered to 0.5 ps . temperature 
 was increased linearly from 300 to 700 k over 1 ns , held constant 
 at 700 k for 1 ns , and then reduced linearly to 300 k over 2 ns . the 
 nmr restraints were lowered to 80% of their strength for the first 
 1.5 ns , and then linearly increased to 100% over the next 500 ps , 
 and remained at 100% strength for the duration of the simulation . the new average structures were used to start a second series of 
 250 ns unrestrained production runs . production md simulation runs 
 were performed for every model in the new ensemble , with constant 
 pressure and volume . temperature was set to 300 k and regulated using 
 the langevin algorithm with a collision frequency of 5 ps . bonds to hydrogen were constrained 
 using the shake algorithm , allowing use of a 2 fs time step . we did not rerefine the nmr structures 
 where mg was 
 present , since model 1 from 1yn2 was stable in the 10 initial 250 ns simulations . to 
 separate the representative structures present in the bimodal distribution 
 of mg - bound slv rna , kmeans clustering was used to group 
 structures into two clusters from all snapshots with an rmsd to the 
 mgbound reference below 2.5  ( supporting script 5 ) . clustering was performed on the heavy atoms of residues 
 u695 to a701 , including the loop and its closing 
 base pair ( supporting script 6 ) . temperature replica exchange md simulations were performed to enhance 
 the sampling space accessible to the mgfree rna in the presence of 
 monovalent ions . model 1 of the mgfree nmr ensemble 1tbk was solvated with 
 tip3p water in an octahedral box . the 
 rna was placed with a buffer of 9  of water , resulting in the 
 addition of 3953 water molecules . sixteen k ions were 
 added to neutralize the system charge and six additional kcl molecules 
 were added to generate an 50 mm excess k concentration . all initial ion positions were randomized with placement at least 
 6  from the rna and at least 4  from each other by swapping 
 positions with random water molecules . joung simulations were carried out using the amber12 and developmental 
 versions of the amber14 suite of programs using the gpu ( cuda ) 
 version of pmemd with spfp ( a mixed single / fixed precision model on 
 the gpu ) . the ff12 force field for nucleic 
 acid simulations was used , which combines ff99 + parmbsc0 modifications 
 + chiol3 modifications for rna . crick restraints on the 
 stem ( residues 690694 and 702706 ) , enhancing sampling 
 of the five loop residues and closing base pair only ( residues 695701 ) , 
 and allowing a smaller solvent box . the restraints were enforced during 
 minimization and equilibration using a force constant of 20 kcal mol  . minimization and equilibration 
 were performed as previously described . for the t - remd simulations , 48 temperatures spanning 277 to 
 400 
 k were carried out in the nvt ensemble using the pmemd.cuda.mpi module of amber 14 . the langevin thermostat 
 was used to regulate temperature with a collision frequency of 5 ps . the particle mesh ewald 
 method , with default parameters , was used to handle electrostatic 
 interactions with a 9  cutoff for direct interactions . ig = 1  option was 
 set to generate random seeds for each restart , avoiding synchronization 
 effects . repartitioning of the solute hydrogen masses to 3.07 au was 
 performed by decreasing the mass on atoms to which the h is bonded 
 by a respective amount , allowing a 4 fs time step . results were plotted using grace or microsoft excel . the sorting 
 of replica trajectories and all analysis grid density analysis was performed for mg ions , and the histogram of the ions was calculated with 
 a grid spacing of 0.5  and normalized by ion density ( supporting script 3 ) . hydrogen bond analysis 
 was performed setting the cations as solvent acceptors and the entire 
 rna as solvent donors , using a distance cutoff of 4.5  ( supporting script 4 ) . the nmr structure of slv in 50 mm nacl found 
 that the canonical u - turn 
 characteristics were absent , although stacking of g697a698c699 was observed . ten 250 ns length - independent 
 md simulations were carried out in 50 mm nacl , starting from model 
 1 of the nmr ensemble ( 1tbk ) , each with a different random starting velocity as 
 well as different randomized ion positions . the all - residue rmsd for 
 each simulation was calculated with reference to the model 1 starting 
 structure ; supporting figure 2 shows that 
 the heavy atom rmsd can be attributed mainly to flexibility seen in 
 the loop region , since the stem rmsd remains consistently around 2.5 
  for the duration of the simulations . the base stacking of the 
 g - a - c triplet was disrupted , and the g697 alpha torsion 
 angle of 116 was not stable ( supporting figure 3 ) . to test the influence of that torsion angle on the 
 loop structure , it was restrained in a separate 100 ns length md simulation 
 that started from model 1 . the resulting trajectory did not show an 
 improvement in the stability of the loop structure , however , and its 
 rmsd from the starting structure was not reduced suggesting that deviation 
 from the extreme alpha angle of 116 was not the root cause of 
 instability of the loop structure ( supporting figure 4 ) . overall , the resulting md simulation structures 
 were more compact with respect to the much extended initial nmr ensemble . supporting figure 5 shows decreased major groove 
 distances across the loop and stem compared to the same distances 
 in the nmr ensemble . we hypothesize this is due to the inclusion of 
 an electrostatic component in the force field that was absent during 
 the original refinement . to better understand the structure , we rerefined 
 the original mgfree ( 1tbk ) structure using the nmr data in the presence of explicit solvent , 
 counterions , the excess ion environment , and with a proper treatment 
 of the electrostatics . the nmr structure was originally refined in vacuo with xplor using a simplified version of the charmm force field 
 that omitted the attractive van der waals and the proper electrostatic 
 contributions . we have previously seen 
 that using explicit water and ions , together with more complete force 
 field parameters ( that is , with electrostatic and attractive van der 
 waals force field components included in addition to explicit solvent 
 and counterions ) can improve refinement of structures from nmr data . each of the 10 structures in the mgfree nmr ensemble was rerefined 
 as described in the experimental methods . 
 from each of the final 6 ns of restrained simulations , which started 
 from each of the rerefined structures , a new average structure was 
 calculated . deviations from the experimental restraints 
 were calculated using the sander module in amber ( employing the pencut 
 = 1 input flag for analyzing all restraints ) , and are shown 
 in table 1 . the members 
 of the rerefined ensemble are in good agreement with the experimental 
 distance and torsion restraints , and are more similar to their minimized 
 average structure , suggesting that the structure determination was 
 more consistent . the rerefined ensemble contains two main conformations , 
 shown in figure 2 , 
 bottom right , and described in table 2 . conformation 1 is the dominant conformation , adopted 
 by 70% of the final ensemble , and conformation 2 is the less dominant 
 conformation , adopted by 30% of the final ensemble ( figure 2 , highlighted in green ) . the 
 characteristic u - turn features of the original nmr ensemble , the original 
 mgbound nmr ensemble ( 1yn2 ) , the rerefined ensemble , and conformations 1 and 
 2 are reported in table 3 . conformation 1 , with its g697  torsion angle 
 of 139 , is more comparable to the original nmr structure with 
 its g697  torsion angle of 116 , while 
 conformation 2 adopts a more canonical u - turn  torsion angle 
 of 154 , closer to the mgbound ensemble . for perspective , most 
 canonical u - turns adopt  torsion angle values from 160 
 to 170 ( supporting table 1 ) . an exception 
 is hiv-1 a - rich hairpin loop which adopts an average  torsion 
 value of 135 , which is very similar to the rerefined value of 
 conformation 1 . another characteristic feature of u - turns is 
 the hydrogen bond 
 between u 2 oh and r n7 on the minor groove side of the loop 
 ( refer to figure 1c , d ) . 
 in slv , this hydrogen bond between u696 2 oh and a698 n7 is 
 present in conformation 2 at an average heavy atom distance of 3.1 
  , which is within error bars of the original mgbound nmr ensemble . 
 while conformation 1 maintains a similar geometry , it samples a longer 
 average distance of 3.8  , which is more similar to the original 
 mgfree nmr ensemble . another attribute of u - turns is the stacking 
 of u with the 5 phosphate group of r. in slv , both with and 
 without mg present , these u696 and a698 5 phosphate 
 distances are 4.2  , and remain this average distance in the 
 rerefined ensemble . however , conformation 1 consistently adopts lower 
 distances of 4.0  at which stacking interactions may occur , 
 similar to the mgbound nmr ensemble . conformation 2 adopts longer 
 distances of 5.2  at which no stacking would occur , as in the 
 mgfree original nmr ensemble . what we conclude from this rerefinement 
 is that the mgfree slv rna can adopt some canonical u - turn characteristics 
 and equally fulfill the experimental nmr restraints . top : original nmr ensemble ( left , 
 models 110 from pdb 1tbk ) and minimized average structures from the rerefined 
 nmr ensemble ( right ) . bottom : u - turn loop residues in the original ensemble 
 ( left ) and after rerefinement ( right ) . three of the rerefined average minimized structures 
 deviate from the rest of the ensemble and are highlighted green . recalculated using the sander program 
 in amber12 . to understand the differences 
 in dynamics of slv when mg ions are either present or 
 absent , we ran four sets of independent simulations , starting from 
 mgbound or mgfree slv rna structures in the presence of monovalent 
 or divalent ions . we started from model 1 of the mgbound nmr ensemble 
 ( mgbound starting structure ) or each model of the rerefined mgfree 
 ensemble ( mgfree starting structure ) , and ran 10 250300 ns 
 length md simulations in either 40 mm mgcl2 or 50 mm nacl . the rmsd correlation plots shown in figure 3 measure the deviation from the mgbound loop 
 ( x - axis ) and mgfree loop ( y - axis ) , 
 and are colored by the number of structures adopting a particular 
 rmsd value . below each correlation plot is a 1-dimensional histogram 
 which shows each of the 10 simulations rmsd to the mgbound 
 loop ( x - axis ) . from the 1-dimensional histograms 
 it is apparent that there is a bimodal distribution of the slv rna 
 structures in the presence of 40 mm mgcl2 with peaks centered 
 on 1.3 and 2.0  . in contrast , there is a single broad distribution 
 of slv rmsds to the mgbound loop structures in the presence of 50 
 mm nacl centered on 2.0  . the slv rna prefers the same conformational 
 minima when the ion environment is the same , despite the conformation 
 of the starting structure . interestingly , the 2-dimensional correlation 
 plots show that in the presence of mg , the population 
 of lower 1.3  rmsd structures are directly accessible only from 
 the 2.0  structures . in this way , the 2.0  structure is 
 a gatekeeper , or an intermediate structure in the path to the mg bound loop . in the absence of mg , the simulations 
 show the loop populates many structures , but this gatekeeper structure 
 represents the most populated structure . however , in the presence 
 of mg , the loop populates a more restricted set of structures . the gatekeeper structure is still populated , but the rna is able to 
 form a structure more similar to the mgbound reference with the mg ions present . the rna is sampling structures that are close 
 to the folded loop structure ( i.e. , the gatekeeper structure at 2.0 
  rmsd to the mgbound reference ) , but mg is required 
 to stabilize the u - turn in its final form ( i.e. , at 1.3  rmsd 
 to the mgbound reference ) . top : 
 2-dimensional histograms of loop rmsd to the mgbound ( x - axis ) and mgfree ( y - axis ) reference structures , 
 defined here as model 1 of 1yn2 and the average structure of the rerefined ensemble , 
 respectively . data collected from independent 250300 ns md simulations 
 for each of 10 systems . each system started from the mgbound reference 
 or each member of the rerefined mgfree ensemble ( labeled mgbound or 
 mgfree , respectively ) , in a divalent ion environment ( + 40 mm mgcl2 ) or monovalent ion environment ( + 50 mm nacl ) . bottom : 1-dimensional 
 histogram graphs of the loop rmsd to the mgbound reference structure 
 are close - ups of the 1.0 to 3.0  region of the larger plot . we wanted to compare the 2.0  
 representative structures found 
 in the presence and absence of mg to the low 1.3  
 representative structure preferred , and only seen , when mg is present . to do so we only considered structures with rmsds to 
 the mgbound loop below 2.5  , and split each of these structure 
 sets by rmsd similarity into two clusters using the kmeans algorithm 
 in cpptraj ( supporting scripts 3 and 4 ) . figure 4a shows the rmsd 
 from the mgbound loop distribution for the top cluster from divalent 
 ion simulations ( black ) , representing the 1.3  structure , the 
 top cluster from monovalent ion simulations ( blue ) , representing the 
 2.0  gatekeeper structure , and the second cluster from divalent 
 ion simulations ( red ) , representing the 2.0  gatekeeper structure 
 observed in simulations with mg present . the distance 
 across the major groove of the loop ( figure 4b ) , represented by a phosphate  phosphate 
 distance of residues u695 to a698 , shows a shorter 
 distance is sampled by the top divalent ion cluster , and slightly 
 longer distances are sampled by the second cluster and the top cluster 
 in the absence of mg . similarly , the radius of gyration 
 ( figure 4c ) of the 
 unr u - turn residues is smaller in the top divalent ion cluster , indicating 
 a tighter u - turn in the presence of mg . the representative structures 
 for the top clusters in divalent and monovalent ions are shown overlapped 
 in figure 4d , and again 
 demonstrate the more compact u - turn of the loop in the presence of 
 divalent ions . the representative gatekeeper structures from 2.0  
 are overlapped in figure 4e , and show a very similar looser u - turn structure . the overall similarity between these structures table 4 shows the results of this analysis 
 for each structure in the three clusters compared to the original 
 mgbound nmr ensemble . though the top cluster from the divalent ion 
 environment showed the best fit to experiment with an 0.01  
 noe deviation , the second divalent ion cluster and top monovalent 
 ion cluster s deviation of 0.02  were still remarkably 
 small and very similar to each other . a detailed analysis of deviations 
 in the loop noes can be found in supporting table 
 2 , and shows that different noes are violated by each cluster . 
 specifically , the less compact gatekeeper loop structure had violations 
 involving residues u696 , g697 , and a698 , again reflecting the less tight u - turn conformation . differences 
 in top clusters populated in monovalent and divalent 
 ion environments . normalized histograms of ( a ) loop rmsd to mgbound 
 reference structure , ( b ) major groove distance , measured from phosphate 
 atom of residue u695 to phosphate atom of residue a698 , ( c ) radius of gyration of residues u696 , g697 , and a698 phosphate groups center of 
 mass . overlap of representative structures loop regions from 
 ( d ) top clusters from divalent ( black ) and monovalent ( blue ) ion simulations , 
 ( e ) top cluster of monovalent ion simulations ( blue ) and second most 
 populated cluster of divalent ion simulations ( red ) ( gatekeeper structures ) . density - based 
 grid analysis was performed on each cluster of the 
 divalent ion simulations to understand the different influence mg had on the more compact and less compact u - turn . the localization 
 of the top 10% of mg ion density is reported in figure 5 and table 5 . figure 5 shows a significant amount of density coordinating 
 the phosphates of the unr motif residues . the association of mg in this region explains the ability of the u - turn to preferentially 
 adopt compact structures , since charged phosphate groups are shielded 
 from each other by the localized charge density of the mg ion . interestingly , the localization of associated mg did not change in the second cluster from divalent ion simulations 
 ( gatekeeper structure ) , despite the less - compact nature of the u - turn 
 ( supporting figure 6 ) . what did change 
 was the percent occupancy , or residence time , of the associated mg , which was far lower for most residues in the second less 
 compact structure ( and is reported in table 5 ) . we hypothesize that this is a result of 
 imperfect clustering , and is a combination of mg ions 
 binding and leaving rather than remaining bound for any length of 
 time . table 5 also 
 identifies that the majority of density is localized in previously 
 described binding sites 1 and 3 . in the 
 simulations starting from mgfree + 40 mm mgcl2 , mg binding in sites 1 and 3 correlates with rna sampling the 
 mgbound conformation , while binding in sites 2 and 4 does not ( supporting figure 7 ) . supporting table 3 details the percent occupancy of all atoms considered 
 part of binding sites 14 , and all of these sites have nonzero 
 occupancy in the simulations . additionally , 
 this table shows the average distance between mg ions 
 and their rna hydrogen - bond acceptors is around 4.0  in each 
 case , indicating that the mg ions associate with the 
 rna through interactions with first shell water molecules and do not 
 directly chelate rna phosphate oxygen atoms . localization of top 10% 
 of mg ion density ( shown in 
 green ) in the top cluster from the divalent ion environment md simulations . to gain insight into the motion the slv rna undergoes 
 to adopt 
 its characteristic conformations in the presence and absence of mg , principal component ( pc ) analysis was performed on three 
 trajectories : the combined 10 simulations in the presence of monovalent 
 ions , the combined 10 simulations in the presence of divalent ions , 
 and the 300 k replica from temperature replica exchange molecular 
 dynamics ( t - remd ) . the t - remd was run starting from the rerefined 
 mgfree structures in the presence of 50 mm kcl , without mg ions . because of the enhanced sampling exchanges performed in t - remd , 
 the conformational space sampled was much larger than the 10 noninteracting 
 simulations combined . pc projections were calculated for the combined 
 trajectories of all systems , and each individual system was independently 
 projected along the top two pcs , shown in figure 6 . the lowest frequency motions described by the pcs are considered 
 relevant to function , with the higher frequency modes describing more 
 local fluctuations . it is apparent that 
 the pc space sampled by the divalent ion simulations ( black ) is more 
 limited than the space sampled by an equivalent amount of simulation 
 with monovalent ions ( red ) . the t - remd samples a much wider range 
 of pc space ( blue ) than either set of simulations . additionally , it 
 is important that the pc space sampled by the divalent ion simulations 
 overlaps with that sampled by the monovalent ion simulations . this 
 indicates that the slv rna is not sampling new conformational space 
 or dynamics in the presence of mg ions , but rather that 
 mg ions serve to limit the dynamics and conformations 
 adopted by the slv rna loop . ten md simulations starting from each member of the rerefined 
 ensemble , mgfree + 50 mm nacl ( red ) , ten md simulations starting from 
 mgbound + 40 mm mgcl2 ( black ) , and the 300 k replica from 
 t - remd simulations of mgfree + 50 mm kcl were combined and common principal 
 components were calculated . the pc projection along modes 1 and 2 
 for each set of simulations are shown above , overlapped , as well as 
 individually . the pc projections for divalent ions ( black ) overlap 
 well with the two sets of simulations performed in a monovalent ion 
 environment ( red and blue ) . the u700 base has been proposed to play 
 a significant role in the dynamics of slv rna . in the presence of mg , the base contributes a phosphate 
 group to a mg binding site , increasing the enthalpic 
 stability of the mgbound slv rna . here specifically , figure 5 shows mg density localized around both 
 the 5 and 3 phosphate groups of the u700 backbone and table 5 and supporting table 3 contain the measured 
 percent occupancies ( note that the 3 phosphate of u700 is equivalent to the 5 phosphate of a701 and 
 is represented as such in the analysis ) . it was also suggested that 
 the u700 base could increase the dynamics of the unbound 
 loop , providing an entropic advantage for sli recognition.figure 7 shows the rms fluctuations for each residue of slv rna in 
 the presence and absence of mg . the highest fluctuations 
 in the absence of mg ions can be seen in residue u700 , and the largest change upon mg association 
 is seen in the same residue . per residue rms fluctuations of slv with monovalent 
 or divalent 
 ions . the rms fluctuations per residue for 10 md simulations of mgfree 
 + 50 mm nacl ( red ) , 10 md simulations of mgbound + 40 mm mgcl2 ( black ) are shown . the 
 varkud satellite ribozyme slv participates in a tertiary structure 
 interaction that aligns the catalytic site of the rna with its intramolecular 
 cleavage site . in the isolated ribozyme , slv forms a kissing complex 
 with the loop of sli , anchoring sli to the ribozyme body , so the properties 
 of slv that allow its three gac nucleotides to form base pairs with 
 sli are essential for a productive interaction . the nmr solution structure 
 of slv was solved based on interpretations of the nmr observables 
 measured with and without divalent ions , and here we revisit those 
 structures with md simulations to describe its molecular processes 
 in more detail . rerefinement of the slv structure ensemble with 
 a more complete 
 description of the nmr solution environment ( including 50 
 mm excess nacl ) suggested that the structures separate into two main 
 loop conformations , each of which equally fulfill the experimental 
 nmr restraints . each conformation adopts some characteristics 
 of the mgbound nmr ensemble , as shown in table 3 , though a single conformation does not adopt 
 all representative u - turn characteristics . md simulations starting 
 from each solution nmr structure ( mgfree 
 and mgbound ) were performed in both a divalent and monovalent ion 
 environment . despite the starting structure , the simulations with 
 divalent ions populated a bimodal distribution of low rmsds to the 
 mgbound loop , with peaks centering around 1.3 and 2.0  , and 
 the simulations with monovalent ions populated a peak at 2.0  
 rmsd from the mgbound loop and did not sample lower rmsd structures . analysis of the representative clusters in the presence of mg and in a monovalent ion environment show that mg binding is required for access to the lower 1.3  rmsd . additionally , 
 the similarity of the monovalent ion cluster and the second cluster 
 of the divalent ion simulations tell us that this structure acts as 
 a gatekeeper for accessing the final mg - bound structure . though the subtle differences in populated structures are an influence 
 of mg binding , analysis of the overall dynamics using 
 principal components shows that the low frequency modes of motion 
 for the slv rna in the presence of divalent ions is a subset of the 
 projection seen for monovalent ions . instead of shifting the slv rna 
 to a previously unseen structure , mg ions instead limit 
 the conformational ensemble and stabilize loop characteristics which 
 are already present in the monovalent ion ensemble . molecular 
 dynamics simulations have traditionally struggled to 
 adequately describe rna structure and dynamics , since they remain 
 extremely sensitive to solution environment and subtle force field 
 parameters that guide nonbonded interactions . here , we present evidence 
 which both qualitatively and quantitatively matches experiment . our 
 improved description of the ion environment adequately reproduces 
 the subtle conformational shift seen in multiple experiments of slv 
 rna . using molecular dynamics simulations of slv 
 rna in the presence 
 of divalent and monovalent ions , we are able to confirm experimental 
 hypotheses on an all - atom level . we show that the u - turn prefers a 
 more compact structure in the presence of mg , effectively 
 neutralizing the electronegative charges of the loop phosphate groups . we confirm the proposed role of the u700 base as a contributor 
 to mg binding site , and as a means to increase entropy 
 of the unbound loop ( and therefore conformational search space for 
 its substrate sli ) in a monovalent ion environment . conformation in the absence of mg , which is a conformational 
 substate leading to mg binding and formation of the tight 
 u - turn structure .
the 
 varkud satellite rna contains a self - cleaving ribozyme that 
 has been shown to function independently of its surroundings . 
 this 
 160 nucleotide ribozyme adopts a catalytically active tertiary structure 
 that includes a kissing hairpin complex formed by stem - loop i and 
 stem - loop v ( slv ) . 
 the five - nucleotide 5-rugacu loop of the 
 isolated slv has been shown to adopt a mg2 + -dependent u - turn 
 structure by solution nmr . 
 this u - turn hairpin is examined here by 
 molecular dynamics simulations in the presence of monovalent and divalent 
 ions . 
 simulations confirm on an all - atom level the hypotheses for 
 the role of the mg2 + ions in stabilizing the loop , as well 
 as the role of the solvent exposed u700 base . 
 additionally , 
 these simulations suggest the mg2 + -free stem - loop adopts 
 a wide range of structures , including energetically favorable structures 
 similar to the mg2 + -bound loop structure . 
 we propose this 
 structure is a  gatekeeper  or precursor to mg2 + binding when those ions are present .