the pentatricopeptide repeat ( ppr ) superfamily ( 1 ) is characterized by a degenerate 35 amino acid repeat and is specific to eukaryotes although most numerous and varied in the plant kingdom ( 2 ) , with over 400 identified in arabidopsis thaliana ( 3 ) .
ppr proteins function in rna stabilization ( 46 ) , editing ( 7 ) , maturation and post - transcriptional modifications ( 8,9 ) and are essential for organelle biogenesis ( 3 ) and translational control ( 1014 ) .
mutations can result in cytoplasmic male sterility ( 15,16 ) , impaired seed development ( 17 ) and diverse embryonic defects ( 18 ) .
ppr proteins can be broadly divided into two families , those consisting of tandem arrays of canonical p - class motifs and those composed of repeats of triplets of p- , l- and s - class motifs ( 3 ) .
regardless of repeat class , conservation of key amino acids from each motif has been correlated with the sequence of known rna targets to establish a statistically and experimentally validated predictive code for rna recognition by ppr proteins ( 1921 ) .
this code is consistent with a modular interaction where a base is coordinated by the sixth residue of one ppr repeat , and the first residue of the next repeat
. such a modular , predictable , mode of single - stranded rna binding makes ppr proteins a potential scaffold for the design of biotechnologically useful proteins ( 22,23 ) .
predictions of an -solenoid tertiary structure for ppr tracts ( 1,24 ) have been confirmed recently by a number of crystal structures including a segment of the mitochondrial rna polymerase ( 25 ) , arabidopsis proteinaceous ribonuclease p ( 26 ) , the p - class ppr protein tha8l ( 27 ) and a synthetic consensus p - class ppr protein ( 28 ) .
crystal structures of two ppr proteins , brachypodium tha8 ( 29 ) and zea mays ppr10 ( zmppr10 ) ( 30 ) , in complex with rna have partially confirmed the modular rna - binding mode , but revealed non - modular and idiosyncratic interactions that had not been anticipated based on prior data .
zmppr10 localizes to plastids where it binds cis - elements of two polycistronic transcripts ( 6,13 ) , the minimal binding sites identified as 17 and 18 nucleotide tracts in the atpi - atph and psaj - rpl33 intercistronic regions .
ppr10 binding was shown to stabilize the rna in vivo , protecting the transcripts from exoribonucleases in either direction , and to activate mrna translation by remodeling local rna structure ( 6 ) .
analytical ultracentrifugation experiments unambiguously demonstrated the association of ppr10 monomers into dimers at high concentration and dissociation of ppr10 dimers into monomers on binding an oligonucleotide representing atph rna , resulting in a 1:1 protein : rna stoichiometry ( 19 ) , with a dissociation constant that has been measured at ca . 1 nm ( 6 ) .
in the recent crystal structure of zmppr10 in complex with an 18 nucleotide rna fragment of psaj ( pdb 4m59 ( 30 ) ) , the protomer consists of a continuous array of 20\documentclass[12pt]{minimal } 
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 } { } $ \frac{1}{2}$\end{document } p - class ppr repeats forming a right - handed superhelix .
a dimer is generated by the association of two anti - parallel molecules , and one rna molecule is bound at each end of the dimer by elements of both monomers .
notably , reported attempts to crystallize ppr10 with atph resulted in apparent dissociation of the dimer and failure to crystallize , suggesting the possibility of a different , higher - affinity , mode of rna binding compared to psaj , for which a dissociation constant of > 200 m has been measured ( 13 ) .
here we use size - exclusion chromatography - coupled synchrotron small - angle x - ray scattering ( saxs ) ( 31 ) to produce clear structural evidence for a monomeric ppr10:atph complex in solution , demonstrating a potential structural rearrangement with respect to the ppr10:psaj crystal structure , that is more consistent with the proposed ppr  rna recognition code and with other prior data concerning ppr10rna interactions .
the zmppr10:pmal - tev plasmid generated as described previously ( 13 ) was used to transform e. coli rosetta 2 ( de3 ) ( novagen ) .
cells were grown in 2yt media containing 1% ( w / v ) d - glucose , 50 mm tris - hcl at ph 7.5 with kanamycin at 50 g ml and chloramphenicol at 50 g ml .
the culture was grown at 37c to an optical density ( 600 nm ) of 0.6 and then cooled on ice for 5 min .
expression was then induced with 1 mm isopropyl -d-1-thiogalactopyranoside and the culture shaken overnight at 16c for protein expression .
the bacterial pellet was resuspended in buffer a ( 50 mm tris - hcl at ph 8.0 , 500 mm kcl , 10% ( v / v ) glycerol , 1 mm dithiothreitol ) supplemented with 0.13 mm phenylmethanesulfonyl fluoride ( roche ) , 1 mini complete protease inhibitor tablet ( roche ) , 0.5 l benzonase ( sigma - aldrich ) and 1 mg ml lysozyme , and lysed under high pressure using a emulsiflex c5 homogenizer ( avestin ) .
lysate supernatant was batch loaded onto amylose resin ( ge healthcare ) and left at 4c for 1 h with agitation .
the column was washed with buffer a , and fusion protein eluted with buffer a supplemented with 50 mm maltose .
fusion protein was cleaved overnight at 4c with tobacco etch virus protease and then subjected to size - exclusion chromatography ( biologic duoflow , bio - rad ) with a hiload 16/60 superdex 200 prep grade column ( ge healthcare ) developed in buffer a. peak fractions were confirmed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( nupagenovex 412% , bis - tris gel , invitrogen ) stained with coomassie brilliant blue .
purified proteins were concentrated in a vivaspin 20 ( mwco 30 000 ) centrifugal concentrator ( ge ) and quantified by absorption at 280 nm ( nanodrop lite , thermoscientific ) .
ppr10:atph complex was prepared by briefly incubating a 1:1 molar ratio of purified protein with rna ( 5-guauccuuaaccauuuc-3 [ idt ] ) .
purified ppr10 protein at a concentration of 0.1 mg ml was dialysed into 10 mm potassium phosphate at ph 8.0 , 100 mm kf , and analysed at 20c in a 1 mm path length quarz cuvette on a jasco j-810 spectropolarimeter .
measurements from 260 to 185 nm with wavelength steps of 1 nm were conducted in triplicate to facilitate the generation of error bars denoting standard deviation .
estimations of -helix content were made using characteristic signals at 208 and 222 nm:%208 = [ ( |[]208|4000 ) / ( 33 0004000 ) ] x 100 and % 222 = [ ( |[]222|3000 ) / ( 36 0003000 ) ] x 100 ( 32,33 ) .
saxs measurements were conducted at the saxs / waxs beamline of the australian synchrotron with continuous data collection on a 1 m pilatus detector ( 34 ) .
sec - sy - saxs was controlled by a shimadzu hplc system . for the ppr10:atph complex ,
scattering data were collected from 50 l of a 6 mg ml sample loaded onto a superdex200 precision column ( ge healthcare ) at 0.1 ml min in 50 mm tris - hcl at ph 7.3 , 250 mm nacl , 10% glycerol , 5 mm  -mercaptoethanol . for the mbp - ppr10:atph complex
, 90 l of a 6 mg ml sample was run through the precision column at 0.5 ml min in buffer a. for experiments where timed shutter opening was critical , the delay between the fplc uv detector and x - ray detector was calibrated using glucose isomerase solution as a standard .
data were corrected for fouling by aggregates while the shutter was open , by using linear interpolation of background from averaged frames prior to the peak to averaged frames after the peak , in an analogous approach to that used in us - somo ( 35 ) . a high - q cutoff of 0.21 was used due to increasing noise beyond this resolution .
the radius of gyration ( rg ) , maximum dimension ( dmax ) and p(r ) distribution plots of the samples were determined with the atsas software ( 36 ) using primus ( 37 ) and gnom ( 38 ) respectively .
however , we chose to manually increase dmax as it was underestimated judging by the appearance of the p(r ) plot ( discussed below ) .
the starting model was derived from a single chain of ppr10 extracted from pdb 4m59 ( 30 ) , broken into seven 
rna was included in the domain definition by placing the appropriate trinucleotide adjacent to the predicted rna - binding residues of each domain .
for the mbp - ppr10 data , pdb entry 1mbp was used as an additional protein domain .
two distance constraints per domain were added to ensure physically reasonable modeling , as the ppr motifs in ppr10 form a continuous array .
crysol ( 41 ) and moleman2 ( 42 ) were used to evaluate model quality by quantifying how representative the atomic models are of the data and p(r ) distribution plots .
the zmppr10:pmal - tev plasmid generated as described previously ( 13 ) was used to transform e. coli rosetta 2 ( de3 ) ( novagen ) .
cells were grown in 2yt media containing 1% ( w / v ) d - glucose , 50 mm tris - hcl at ph 7.5 with kanamycin at 50 g ml and chloramphenicol at 50 g ml .
the culture was grown at 37c to an optical density ( 600 nm ) of 0.6 and then cooled on ice for 5 min .
expression was then induced with 1 mm isopropyl -d-1-thiogalactopyranoside and the culture shaken overnight at 16c for protein expression .
the bacterial pellet was resuspended in buffer a ( 50 mm tris - hcl at ph 8.0 , 500 mm kcl , 10% ( v / v ) glycerol , 1 mm dithiothreitol ) supplemented with 0.13 mm phenylmethanesulfonyl fluoride ( roche ) , 1 mini complete protease inhibitor tablet ( roche ) , 0.5 l benzonase ( sigma - aldrich ) and 1 mg ml lysozyme , and lysed under high pressure using a emulsiflex c5 homogenizer ( avestin ) .
lysate supernatant was batch loaded onto amylose resin ( ge healthcare ) and left at 4c for 1 h with agitation .
the column was washed with buffer a , and fusion protein eluted with buffer a supplemented with 50 mm maltose .
fusion protein was cleaved overnight at 4c with tobacco etch virus protease and then subjected to size - exclusion chromatography ( biologic duoflow , bio - rad ) with a hiload 16/60 superdex 200 prep grade column ( ge healthcare ) developed in buffer a. peak fractions were confirmed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( nupagenovex 412% , bis - tris gel , invitrogen ) stained with coomassie brilliant blue .
purified proteins were concentrated in a vivaspin 20 ( mwco 30 000 ) centrifugal concentrator ( ge ) and quantified by absorption at 280 nm ( nanodrop lite , thermoscientific ) .
ppr10:atph complex was prepared by briefly incubating a 1:1 molar ratio of purified protein with rna ( 5-guauccuuaaccauuuc-3 [ idt ] ) .
purified ppr10 protein at a concentration of 0.1 mg ml was dialysed into 10 mm potassium phosphate at ph 8.0 , 100 mm kf , and analysed at 20c in a 1 mm path length quarz cuvette on a jasco j-810 spectropolarimeter .
measurements from 260 to 185 nm with wavelength steps of 1 nm were conducted in triplicate to facilitate the generation of error bars denoting standard deviation .
estimations of -helix content were made using characteristic signals at 208 and 222 nm:%208 = [ ( |[]208|4000 ) / ( 33 0004000 ) ] x 100 and % 222 = [ ( |[]222|3000 ) / ( 36 0003000 ) ] x 100 ( 32,33 ) .
saxs measurements were conducted at the saxs / waxs beamline of the australian synchrotron with continuous data collection on a 1 m pilatus detector ( 34 ) .
sec - sy - saxs was controlled by a shimadzu hplc system . for the ppr10:atph complex ,
scattering data were collected from 50 l of a 6 mg ml sample loaded onto a superdex200 precision column ( ge healthcare ) at 0.1 ml min in 50 mm tris - hcl at ph 7.3 , 250 mm nacl , 10% glycerol , 5 mm  -mercaptoethanol . for the mbp - ppr10:atph complex , 90 l of a 6 mg ml sample was run through the precision column at 0.5 ml min in buffer a. for experiments where timed shutter opening was critical , the delay between the fplc uv detector and x - ray detector was calibrated using glucose isomerase solution as a standard .
data were corrected for fouling by aggregates while the shutter was open , by using linear interpolation of background from averaged frames prior to the peak to averaged frames after the peak , in an analogous approach to that used in us - somo ( 35 ) .
a high - q cutoff of 0.21 was used due to increasing noise beyond this resolution .
the radius of gyration ( rg ) , maximum dimension ( dmax ) and p(r ) distribution plots of the samples were determined with the atsas software ( 36 ) using primus ( 37 ) and gnom ( 38 ) respectively .
however , we chose to manually increase dmax as it was underestimated judging by the appearance of the p(r ) plot ( discussed below ) .
the starting model was derived from a single chain of ppr10 extracted from pdb 4m59 ( 30 ) , broken into seven 
rna was included in the domain definition by placing the appropriate trinucleotide adjacent to the predicted rna - binding residues of each domain .
for the mbp - ppr10 data , pdb entry 1mbp was used as an additional protein domain .
two distance constraints per domain were added to ensure physically reasonable modeling , as the ppr motifs in ppr10 form a continuous array .
crysol ( 41 ) and moleman2 ( 42 ) were used to evaluate model quality by quantifying how representative the atomic models are of the data and p(r ) distribution plots .
we have generated a plausible model of the solution structure of zmppr10 in complex with the 17 nucleotide minimal binding site of the maize atph transcript informed by a combination of saxs data , circular dichroism spectroscopy , and pre - existing bioinformatic , biochemical and crystallographic data .
the secondary structure content of ppr10 was estimated to be predominantly -helical ( 84% ) from circular dichroism as expected for the anti - parallel -helical topology of a ppr motif , and comparable with calculations based on the crystal structures ( 71% -helix ) .
preliminary saxs measurements on ppr10 alone and after incubation with atph rna yielded complex uninterpretable scattering profiles due to significant aggregation .
we therefore employed size - exclusion chromatography - coupled synchrotron small - angle x - ray scattering ( sec - sy - saxs ; figure 1 ) .
this method allowed us to separate the various complexes inline , identify their content from their a260/a280 ratios and examine the solution structure of specific sec fractions ( figure 1a ) . a significant challenge to this method
was encountered due to the earliest eluting aggregated proteins adhering to the sample capillary at the point of x - ray exposure , thus tainting all further images . by carefully timing the opening of the x - ray shutter to only measure regions of interest and appropriate background segments it was possible to measure high quality saxs on the protein : rna complex .
scattering data were obtained for a monodisperse peak with an a260/a280 ratio and molecular size commensurate with a monomeric ppr10:atph complex ( figure 1b ) .
( a ) size exclusion chromatography trace of ppr10:atph showing peaks corresponding to oligomers , ppr10:atph complex and unbound atph rna .
black bars indicate the part of the chromatogram for which saxs was collected by opening the beamline shutter .
the grey circle indicates position where the first data frame was recorded , and the bar , the region corresponding to frames 552710 .
( b ) scattering profiles of the ppr10:atph complex averaged across 20 frame segments , corresponding to the second half of the ppr10:atph peak ( grey bar in ( a ) ) .
( c ) guinier analysis of the low - q scattering data for the five most strongly scattering 20 frame segments indicates a radius of gyration of 36  .
( e ) dimensionless kratky plot is diagnostic of a globular but somewhat extended protein .
maximum dimension , dmax , and porod volume , vp , and molecular mass , mr , determined using gnom .
oligomeric state ( n ) determined from the ratio of mr(vp ) to mr ( theoretical ) .
guinier analysis of the low - q data indicated a consistent radius of gyration ( rg ) close to 36  for the most concentrated fractions ( table 2 , figure 1c ) .
an upturn at low - q became evident in the scattering profile during later fractions , possibly due to adhesion of protein on the capillary ( figure 1b ) , rendering guinier analysis of these lower concentration samples impossible .
thus , data from the peak fraction ( frames 552570 ) were averaged and used for subsequent analysis .
the p(r ) distribution ( figure 1d ) is indicative of a globular structure with an extended tail : an asymmetric distribution with a maximum at 40  with a shoulder at 20  and a steady decline through the longer distances with shoulders at 80 and 110  and a dmax of ca .
a dimensionless kratky plot contains a peak at 1.95 with a height of around 1.2 indicating a somewhat elongated , but generally globular protein ( with reference to ( 43 ) ) that is suitable for porod analysis ( figure 1e ) .
the molecular mass ( mr ) of the scattering particle , determined from the porod invariant ( vp ) , ( 44 ) , corresponds to a monodisperse , 1:1 ppr10:atph complex ( tables 1 and 2 ) . as the monomeric state of the complex as determined by auc , remsa and now saxs contrasts with the reported dimeric crystal structure , we next sought to build a molecular model  taking into account the limitations of data quality  that best reflects the scattering data .
we first examined the crystal structures of zmppr10 in the absence ( 4m57 ) and presence ( 4m59 ) of psaj rna .
initially , we generated candidate models of the observed dimers , and monomers extracted from the crystal structures , and compared their minimized fit with the observed scattering profiles , and compared their distance distributions with the observed p(r ) distribution ( figure 2 ) .
the relatively high  values combined with the lack of common features in the distance distribution ( e.g. a maximum at 60  compared to 40  ) led us to conclude that there were notable differences between the solution structure and the crystallographic models .
bottom row : reconstructed pair distribution plots from the respective models ( solid histogram ) compared with the experimentally derived p(r ) distribution ( black line ) .
( a ) apoppr10 monomer extracted from pdb 4m57 ; ( b ) apoppr10 dimer from pdb 4m57 ; ( c ) ppr10:psaj monomer extracted from pdb m459 ; ( d ) ppr10:psaj dimer from pdb 4m59 ; ( e ) the best - fitting sasref model .
armed with this knowledge we generated rigid body models from ppr10 broken into seven domains , with rna fragments attached , and refined against the scattering data .
highly consistent results were obtained over multiple independent modeling runs for ppr10:atph ( figure 2e ) presented as a monomeric complex .
the best - fitting model is a right - handed superhelical array with the c - terminal region forming a toroid .
however , the pseudosymmetric nature of the ppr10 molecule  a superhelical array  made it impossible to objectively assign either end as the n- or c - terminus .
to address this problem , we repeated the data collection procedure with an n - terminal mbp - ppr10 fusion protein .
scattering data were analysed ( figure 3 ) revealing increases of rg to 61  and dmax to 250  , as expected from addition of an mbp domain .
rigid body modeling of this complex produced a satisfactory solution (  = 0.78 ) for a protein with mbp at the n - terminus and a toroidal arrangement at the c - terminus , thus supporting our model of the untagged protein , which is used in the following comparative analysis .
sec - sy - saxs analysis of mbp - ppr10 fusion protein : atph complex .
( a ) scattering profiles of averaged and background subtracted five - frame region of the peak fraction of mbp - ppr10:atph .
( b ) guinier analysis and ( c)p(r ) distribution for the 215220 averaged scattering profile .
alignment of monomeric ppr10 structures from three different available structural contexts ( a monomer extracted from the apo - ppr10 dimer ( pdb 4m57 ) ; a monomer extracted from the psaj - bound dimer ( pdb 4m59 ) and the monomeric atph - bound solution structure , this work ) reveals two notable regions of structural rearrangement ( figure 4a ) that are also discernable from the scattering profiles and distance distributions ( figure 2 ) .
while the c - terminal region of the apo- and psaj - bound monomers aligns well , the four n - terminal ppr motifs are arranged differently .
conversely , the psaj- and atph - bound conformations only differ significantly around the four c - terminal ppr motifs .
( a ) dynamic structure of a ppr10 monomer : a single ppr10 molecule extracted from the rna - free dimer structure ( 4m57 chain a , red cartoon ) , the ppr10:psaj dimer ( 4m59 chain a , gold ) and the monomeric ppr10:atph solution structure ( this work , blue ) can be superimposed effectively ( rmsd ranging from 7 to 11  including c atoms from most of the structure : black lines link superimposed c atoms between two structures ) .
the n - terminal region ( oriented at the top ) differs between apo- and psaj - bound proteins , while only the c - terminus differs significantly between psaj- and atph - bound proteins .
( b)psaj rna coordination by ppr10 motifs 6 and 7 in the crystal structure ( 4m59 ; chain a orange cartoon ; chain b , beige / gray surface ) .
residues asn284 , asp314 and asn319 , asn349 ( positions 6 and 1 for motifs 6 and 7 respectively ) are known to dictate rna specificity , however asn349 is displaced from rna binding by the dimeric interface .
( c ) the solution structure of ppr10:atph illustrates that the arrangement of the ppr motifs ( colored blue to red from n - terminus ) is conducive to ssrna binding ( black cartoon ) as a monomer ( note : breaks in rna chain are a consequence of the rigid body modeling approach used ) .
this study shows how the coupling of size - exclusion chromatography with synchrotron saxs allows productive study of challenging macromolecular complexes , particularly when studying a well - characterized system with ample orthogonal information , from genomic , biochemical and crystallographic sources .
we have determined that the solution structure of zmppr10 complexed with a fragment of atph is not compatible with the dimeric form observed in the ppr10:psaj crystal structure , thus confirming previous analytical ultracentrifugation analyses .
the nature of recognition of atph and psaj has been well characterized ( 6,13,19 ) , and the ppr 
rna recognition code has been validated biochemically for the n - terminus/5 end of the ppr 
rna duplex by mutating residues asn284 , asp314 and asn319 , asn349 in ppr motifs 6 and 7 , and observing a predictable change in binding specificity ( 19 ) .
the dimeric conformation observed in the psaj - bound ppr10 structure is not fully compatible with this result , as one of the key residues in motif 7 does not engage the rna ( figure 4b ) .
indeed the structure of the helix bearing asn349 is highly disturbed , presenting considerably different positions in the two subunits of the crystal structure : in both cases asn319 is well over 10  distant from the rna .
it seems likely that the presence of the other subunit of the dimer close to this region is a cause of the disruption of this binding site ( figure 4b ) . in the saxs structure ,
the more highly overwound superhelix of the c - terminal region would sterically hinder dimerisation of the protein ( figure 4c ) . in this tight , toroid - like part of the structure there are intramolecular interactions between residues of the c - terminal repeats and repeats earlier in the sequence , which in the dimeric crystal structure are involved in intermolecular interactions instead
. our results are consistent with the 1:1 stoichiometry established by analytical ultracentrifugation for a ppr10-atph complex ( 19 ) .
that a similar architecture would apply to the ppr10-psaj complex in solution is supported by the comigration of ppr10-atph complexes and ppr10-psaj complexes in native gels and by the extensive sequence conservation across the length of the psaj and atph rnas .
a question is raised about whether all of these unambiguously observed configurations can be correct .
the answer may lie in the necessarily convoluted dynamic process that is required for ppr10 to dissociate from a dimeric state to a monomeric state and to bind sequence specifically to rna in a cellular context involving other binding partners and rna secondary structures ( 6 ) .
it is unclear whether the dimeric ppr10:psaj structure observed crystallographically represents an intermediate state in the dynamic process of rna recognition by ppr10 , or if it represents a dead - end complex formed at the high concentrations required for crystallisation . at a late point in our analysis ,
a paper became available that raises further doubt regarding the dimeric nature of the complex ( 45 ) . here
, the authors of the ppr10 crystal structures determine that an n - terminal truncation and a point mutation that were essential for crystallogenesis in fact drive dimerization .
our wild - type ppr10 protein makes a more physiologically relevant model for the interaction between ppr10 and target rna , taking into account the miniscule cellular concentration of ppr10 ( with respect to the micromolar dissociation constant for dimerisation ) and the large excess of  non - specific single - stranded rna . while it remains plausible that dimeric apoppr10 binds to an rna transcript in low - affinity mode causing a structural rearrangement that dissociates the dimer and allows the high - affinity monomeric complex to form , it is likely that in the cell a monomeric ppr10 interacts with bulk single - stranded rna before exchanging partners to achieve high - affinity binding to its target rna motif as a monomeric protein : rna complex similar to the solution structure presented here . despite their well - described modularity and relatively predictable rna - binding specificity
, this example of a native ppr protein reveals a potentially convoluted process of rna binding .
if the promise for ppr proteins to be applied as ssrna - binding tools in biotechnology is to be maximized , a fuller understanding of the detailed structural changes that occur on rna binding is essential .
australian research council ( discovery project dp120102870 to i.d.s . , a.b . , c.s.b . )
pearl technologies ltd ; university of western australia ( scholarship for b.s.g . ) . funding for open access charge : the university of western australia .
 the pentatricopeptide repeat ( ppr ) protein family is a large family of rna - binding proteins that is characterized by tandem arrays of a degenerate 35-amino - acid motif which form an -solenoid structure . 
 ppr proteins influence the editing , splicing , translation and stability of specific rnas in mitochondria and chloroplasts . 
 zea mays ppr10 is amongst the best studied ppr proteins , where sequence - specific binding to two rna transcripts , atph and psaj , has been demonstrated to follow a recognition code where the identity of two amino acids per repeat determines the base - specificity . 
 a recently solved zmppr10:psaj complex crystal structure suggested a homodimeric complex with considerably fewer sequence - specific protein  
 rna contacts than inferred previously . here 
 we describe the solution structure of the zmppr10:atph complex using size - exclusion chromatography - coupled synchrotron small - angle x - ray scattering ( sec - sy - saxs ) . 
 our results support prior evidence that ppr10 binds rna as a monomer , and that it does so in a manner that is commensurate with a canonical and predictable rna - binding mode across much of the rna  protein interface . 
INTRODUCTION
MATERIALS AND METHODS
Protein expression and purification
Circular dichroism analysis
Small-angle X-ray scattering analysis
RESULTS
DISCUSSION
FUNDING