interest in adeno - associated virus ( aav ) stems mostly from its experimental and clinical use as a vector in gene therapy of ( for  example ) hemophilia b and lipoprotein lipase deficiency.1 , 2 , 3 the virus s interactions with host cells are therefore of fundamental interest and of interest as a foundation for development of more specific means of gene delivery .
aavs have single - stranded dna ( ssdna ) genomes that are mostly replaced in gene therapy vectors . the dna is surrounded by a protein shell composed of 60 copies of a capsid protein ( in three variant forms ) , whose structure was first solved by x - ray crystallography for the type species , aav-2 .
functions such as cell attachment , entry , and immune evasion are associated with this capsid shell .
several serotypes , including aav-2 , bind to cell surface heparan sulfate proteoglycan ( hspg ) that was considered , until recently , to serve as the  primary  entry receptor .
structural studies of complexes with the analog heparin were limited to electron microscopy at 2 and 1  nm , respectively,7 , 8 which were insufficient to arbiter between different characterizations of the binding site and the magnitude of glycan - induced conformational changes .
aav - dj complexed with sucrose octasulfate ( sos ) at 5   resolution showed arginines interacting with sulfates , consistent with the earlier 1  nm aav-2/heparin study .
both showed the glycan bound to heparan - binding domains ( hbds ) on the sides of prominent surface spikes , the polymer winding loosely between adjacent hbds related by a 3-fold axis of symmetry .
however , arguably , the aav-2 studies7 , 8 have not been at a resolution sufficient for robust characterization of induced conformational changes , as proposed , motivating the current aav - dj studies at crystallographic - like resolution .
aav - dj is a recombinant variant selected for resistance to human neutralizing sera and for improved liver tropism , which is a chimeric mix of serotypes 2 , 8 , and 9 .
a low - resolution ( 5   ) structure by cryoelectron microscopy ( cryo - em ) , as well as functional properties , indicated gross similarity to aav-2.11 , 12 aav - dj provided somewhat higher - resolution cryo - em data than other aav2-like natural serotypes ( for which it serves as a model ) , likely because it was prepared as a virus - like particle ( vlp ) , devoid of the ssdna in natural virions , thereby offering greater em contrast and superior alignment .
diffraction to 6.5   resolution provided no detail , but interacting arginines could be identified by overlaying the difference map on the 2.6   resolution uncomplexed structure . lying in a depression surrounding the 3-fold axis ,
both gain- and loss - of - function mutants could be created , with stronger or weaker heparin binding and cell transduction , by mixing and matching glycan - binding residues from aav-2 and aav-3b in chimeric mutant viral vectors .
this established that the interactions were additive in glycan binding and less specific than anticipated .
structure - inspired mutation of amino acids in aav-6 has implicated yet another cast of positively charged amino acids in heparin binding , only partly overlapping with the aav-2 site , whereas analogous studies implicated a distinct pocket of mostly polar amino acids in the galactose binding of aav-9 .
although aav-5 , the most divergent in sequence , binds to a different type of glycan , mutation implicates a site similar to that of aav-3b .
structural studies have been complemented by biophysical chemistry , measuring the specificity of binding by surface plasmon resonance ( spr ) with heparinoid libraries or using glycan array technology .
these studies showed modest specificity for different types of heparinoid , their sequences , and sites of sulfation .
they revealed a stepwise dependence of binding on length of oligosaccharide , rationalized in terms of the length needed to fill one hbd site , and the longer length that enables bridging to a symmetry - related site with dramatic increasing in avidity .
thus , studies were needed of a more realistic heparinoid analog than the highly sulfonated sos disaccharide .
fondaparinux is a synthetic five - ring heparinoid , terminated with an o - methyl group , but otherwise identical to sequences prevalent in heparin and heparan sulfate ( figure  1 ) .
it is an anticoagulant marketed by glaxosmithkline under the trade name arixtra , indirectly inhibiting factor xa by binding antithrombin iii .
fondaparinux offered a close analog with similar sulfation to the natural hspg receptor , but without its sequence heterogeneity .
it would be long enough to fill a single hbd site , but not to bridge between symmetric sites , avoiding heterogeneity in the ways that sites might be bridged .
here , the binding of fondaparinux is analyzed and structure is characterized through a cryo - em reconstruction of the aav - dj complex at 2.8   resolution and the unbound virus at 3.5   resolution .
the competition assay measured inhibition by fondaparinux of the binding of aav-2 to a chip coated with heparin ( figure  2 ) .
sensorgrams are similar to those of short heparin fragments ( degree of polymerization [ dp ]  12 ) that can not bridge between symmetry - related sites on the virus .
short oligosaccharides compete poorly with the high - avidity multi - site binding of heparin , precluding quantitative kd determination by curve fitting .
nevertheless , fondaparinux yields a similar reduction in ru as short heparin oligosaccharides ( 2   dp  12 ) .
the effect is dose dependent , with a 5% to 30% reduction in ru for fondaparinux as the concentration is increased from 1 to 20  m . for commensurate inhibition of aav binding to heparin ,
10-fold more fondaparinux is needed than hspg , the native receptor , or the more highly charged sos ( figure  2 ) .
fondaparinux inhibition of aav-2 hela cell transduction is also dose dependent ( figure  2 ) . in the absence of fondaparinux , scaav2-cmv - mcherry at 1.2   10 viral genomes per cell ( vg / cell ) transduces 90% of cells .
concentrations exceeding 10  mm result in loss of cell viability ( data not shown ) presumably because of increased osmolarity .
( transduction and viability were not diminished in sucrose controls at 1030  m [ figure  s1 ] . )
the cryo - em reconstructions of the bound and unbound forms represent the best of several attempts . for the complex , 3.5  
( fourier shell correclation [ fsc]0.143)21 , 22 was achieved with conventional processing and 3.3   with frame - dependent motion correction .
the 2.8   reconstruction was achieved with empirically optimized dose compensation , attenuating high - frequency contributions in later frames , and using 107,454 particles .
detail in the reconstruction allows interpretation at a level hitherto only possible with x - ray crystallography ( figure  3 ) .
the binding site was apparent from a standard ( unweighted )  difference map , calculated by subtracting the reconstruction of the unbound form from that of the complex ( see materials and methods ) .
a 5   limit gave the best difference map , with density enveloping the fondaparinux at 13  ( figure  4 ) .
it is not clear whether the additional features at 4   are real or noise ( figure  4 ) .
thus , refinement of the fondaparinux structure used a conservative density - gradient optimization ( not molecular dynamics ) into the 5   map , using strong restraints to enforce excellent stereochemistry .
thus , the model is representative of a family of possible configurations , whose average corresponds to the observed density .
the maximum difference density at the fondaparinux is at 33 . the next highest peak is 11  ( on a 3-fold axis , where noise is higher ) . resolving the controversy between two lower - resolution aav-2/heparin complexes,7 , 8 the fondaparinux density superimposes on the heparin of the 8.3   complex ( figure  4 ) and on the sos in the aav - dj complex ( figure  4 ) .
the site may not be occupied simultaneously in all 60 symmetry - equivalent locations on the virus capsid .
average ligand occupancy is estimated , two ways , to be equal to or greater than 0.3 .
an estimate of 0.30.4 comes by subtracting progressively more unbound map from complex until the density of ligand and virus are equally strong .
this underestimates occupancy , because the glycan is less constrained than the protein and higher atomic displacements ( b factors ) weaken the density .
a second estimate comes from dual - conformer atomic refinement of the viral protein structure in bound and unbound states ( see later ) , yielding a bound - state occupancy of 0.33 .
this is also an approximation , because it is difficult to distinguish similar bound and unbound protein conformations in the summed density of the experimental reconstruction .
first , it was refined against the highest resolution reconstruction , that of the fondaparinux complex , after seeing that ligand affected only local regions .
starting from the 5   native structure , the atomic model was fit into the 2.8   map , refining by stereochemically restrained real - space torsion angle dynamics .
the 1.0   root - mean - square ( rms ) change in backbone reflected mostly the improved resolution , rather than effects of the ligand ( see later ) . the correlation coefficient improved from 0.61 to 0.84 , when calculated using all grid points within 3.4   of any atom .
aav - dj is a chimeric variant with components of sequence from aav-2 , -8 , and -9 . at one point
, we had hoped to bypass experimental structure through homology modeling using modeler and the then - known structures of aav-2 and -84 , 26 as templates .
the predicted structure was optimized by gas - phase annealing , surrounding a subunit with its nine symmetry - equivalent neighbors to ensure loop conformations consistent with the viral quaternary structure .
the subsequent 5   em structure differed from the prediction by 1.7    ( rms , all atom ) and 1.2   ( c ) .
even though a glycan complex , the 2.8   structure is closer to the prediction , with rms deviations ( rmsds ) of 1.3   ( all atom ) and 1.0   ( c ) , indicating that , overall , a careful homology model can match in quality a 5   experimental structure .
now , with the 2.8   experimental aav - dj structure , we see that variable region 1 ( vr - i ) differs from aav-2 and aav-8 by up to 8 and 11   , and is locally closer ( 1   maximum ) to aav-9 ( table  1 ) .
a homology model with an aav9-like vr - i , built using only aav-8 and aav-9 as templates , is in closer agreement with the experimental structure ( 1.1   , rms all atom , and 0.4   , c ) , but it was only in hindsight that we knew that these and only these templates should be used .
relative to the original aav-2/8-based homology model , side - chain rotamers were changed at 121 ( 23% ) amino acids .
fifty - four solvent waters were modeled peaks into three  peaks with good hydrogen - bonding interactions ( 0.1 waters / amino acid , as expected of an 2.8   x - ray structure ) .
backbone differences from the aav-8/-9 homology model , exceeding the rmsd , are restricted to several loops : near thr456 , with a maximal c shift of 1.8   , gly267 ( 1.5   ) , asn705 ( 1.5   ) , gly548 ( 1.3   ) , gly522 ( 1.3   ) , thr493 ( 1.1   ) , and asn717 ( 1.1   ) ; these regions are key to aav - dj s properties , as discussed later . differences between aav - dj and parental serotypes are summarized in table 1 and figures 5 and s2 .
core regions are highly conserved , with differences limited to surface loops encoding the characteristic functional properties of aav - dj .
conservation of the core is to be expected with overall sequence identities for aav - dj vp1 of 93% for aav-2 , 89% for aav-8 , and 85% for aav-9 .
structural differences are in a subset of the vr loops , previously noted as sites of structural difference and sequence diversity between serotypes .
c differences between aav - dj and any of the parental serotypes exceed 1   at vr - i , -ii , -iii , -iv , -v , and -ix . in vr - i ( residues 262270 ) ,
aav - dj approximates most closely aav-9 ( maximal c deviation  = 1.1   ) , with which it shares the same local sequence , and is quite different from aav-2 and -8 ( maximal c deviations of 8 and 11   ) that have 33% and 67% sequence identity , respectively ( table 1 ) . in vr - iv ( residues 451470 ) , the structure most closely follows aav-8 ( maximal c  deviation  = 2.8   ; 95% local sequence identity ) , deviating by 6  and 8   from aav-2 and aav-9 ( 50% local sequence identity ) .
all of the vrs are surface exposed , and some  have among the weakest density in the aav - dj reconstruction :  vr - iv , vr - ix ( near residue tyr707 ) , vr - i , and vr - ii ( glu329-gly330 ) . as in the crystal structures of the parental serotypes , b factors in these regions refine to high values .
even though some of these regions are somewhat disordered , their structures are distinct . for example
, at gly454-gly455 in vr - iv , aav - dj and aav-8 differ by up to 3   , and both are distinct from aav-2 or aav-9 .
an unbound structure was now obtained by density gradient refinement of this structure into the native 3.5   reconstruction . where the  average  and unbound models differed , the standard difference map was examined . where evidence of conformational change was lacking in the map ,
model differences were assumed to have resulted from lower precision in the 3.5    native reconstruction , and the model was reset to the 2.8   structure . where supported by the difference density , dual conformers were used .
the fully bound conformation was modeled and refined into an occupancy - weighted difference map ( see materials and methods ) designed to subtract contributions to the map from un - liganded protein .
regions far from conformational changes were fixed in their structures refined at 2.8   resolution . finally , dual - conformer refinement allowed the sum of bound and unbound structures to be refined against the full 2.8   data , avoiding limitations that come with the lower - resolution native data .
the increase in degrees of freedom was mitigated by using dual conformers only locally for 10 of 517 residues ( 2% ) , where indicated by the difference map .
the structure was refined with torsion angle - simulated annealing , ignoring close approaches of atoms between bound and unbound states .
because the effects of the glycan upon the virus structure are local and subtle , the 2.8   structure can provide detailed insights into the makings of aav - dj from the parental serotypes under the selection of directed evolution.11 , 28 , 29 in so doing , we compare aav - dj with the x - ray structures of aav-2 , aav-8 , and aav-9 , all at circa 3   resolution .
predominantly , we see that the tertiary loop structure comes from the underlying sequence and is little affected by contacts from a neighboring background of different parental strain .
we also see that the effects on structure of a sequence substitution are local , with no evidence that changes are propagated away from the site .
aav - dj was selected both for transduction of human liver cells and then resistance to neutralization by pooled human antisera ( intravenous immunoglobulin [ ivig ] ) .
liver transduction had selected hybrids from five of eight starting serotypes ( 2 , 4 , 5 , 8 , and 9 ) ; then ivig - escape selected a single hybrid of serotypes 2 , 8 , and 9 .
the most prominent difference between aav - dj and aav-2 is in vr - i at the epitope of neutralizing mouse monoclonal antibody ( mab ) a20.8 , 12 , 31 , 32 this explains the lack of recognition of aav - dj by mab a20 , in spite of 93% capsid sequence identity with aav-2 ( table 1 ; figure  s2 ) .
now , with a 2.8   aav - dj em structure and crystal structures for all parental strains , aav-2 , aav-8 , and aav-9 , detailed comparisons can be made .
germane to the ivig selection , antibody binding sites have been mapped for two parental serotypes ( aav-2 and aav-8 ) and one ( aav-5 ) that was selected out .
mappings have used cryo - em at resolutions of 819   , scanning mutagenesis , and/or peptide scanning.32 , 33 , 34 , 35 , 36 , 37 , 38 structural differences ( > 1.5   ) between aav - dj and natural serotypes represented in the hepatocyte - selected hybrids are predominantly within footprints of neutralizing mab ( figure  5 ) .
conversely , there are differences > 1.5   in all neutralizing mab footprints mapped at nanometer or better resolution .
thus , it is reasonable to propose that predominant structural features selected during the directed evolution of aav - dj have allowed it to escape the binding of neutralizing antibodies .
( it is quite possible that changes selected under ivig pressure have also contributed pleiotropically to altered cell tropism . )
first , the logic implicitly assumes that mouse monoclonals ( like a20 ) are representative of dominant neutralizing antibodies in human serum , which seems plausible , but there is no evidence .
second , the antibody footprints on aav have significant uncertainties , because of sometimes limited em resolution , and known challenges in homology modeling of vrs in antibody loops .
third , the mab footprints cover , in aggregate , a significant fraction of the outer surface .
finally , c differences are just one ( easy ) metric to survey , but sequence differences and side - chain structure will also mediate antibody escape .
there are technical reasons ( see results ) why the fondaparinux occupancy might be underestimated , but weak affinity is consistent with the spr and inhibition assays .
although both fondaparinux and sos are bound by the arginines of the hbd that are implicated in heparin binding,4 , 8 , 10 , 39 binding is weaker than that of heparin .
the lower - bound fondaparinux occupancy 0.3 estimate corresponds to a kd 10  mm upper bound , although binding might actually be stronger .
thus , the ic50 0.7  mm from transduction inhibition ( figure  2 ) is not inconsistent .
however , how can approximate millimolar fondaparinux binding be reconciled with a stronger sub - nanomolar kd for heparin ?
the likely explanation is that glycan polymers achieve strong effective binding ( avidity ) by simultaneously binding to several symmetry - related sites , as no short oligomer can .
the shortest distance between sites on the virus corresponds to 13 saccharide units , so an 17  kda heparin polymer could , in principle , bind at up to six sites . three or more would be required to achieve nanomolar avidity with millimolar affinity at each site .
the ligand was visualized at lower resolution than the viral protein , including the hbd loop and side chains of the binding site .
there are both chemical and technological ( em ) factors that contribute to blurriness , and indeed , ligands are generally less well resolved than protein in em structures .
sos has a higher sulfate content , perhaps leading to tighter electrostatic interactions with binding site arginines .
sos is less flexible and more pseudo - symmetrical , perhaps yielding fewer distinct binding configurations and less disorder .
fondaparinux has sulfation that is more typical of heparin or the high - sulfation regions of heparan sulfate , so its apparently varied configurations may be biologically relevant .
higher order in the surrounding protein suggests a site that is able to bind to the various glycan configurations and sequence elements that aav would encounter within a heparinoid polymer .
it would also explain the observed low specificity , with in  vitro binding to a variety of heparinoids , including some chondroitin sulfates.18 , 19 biological implications are that there might be a variety of cell surface glycans to which each aav serotype might bind , and that there might also be connective tissue or extracellular matrix with glycans that decoy aav away from productive infections .
individual sulfates are not well resolved in the density for fondaparinux , nor were sulfate interactions used as a constraint in model - building .
however , the 2-n - sulfo group of the middle pyranose ring and the 6-o - sulfo group of the fifth ring are both in proximity to the arginines previously implicated in binding , arg587 and arg590 , respectively.4 , 8 , 10 , 39 spr using heparinoid libraries and glycan array binding studies have both indicated that sulfate groups at 2-n and 6-o are more critical than at 2-o.19 , 44 the four modeled fondaparinux sulfates , close to these arginines , superimpose within 24   of sulfates that were resolved in the sos complex .
this modest correspondence is another indication that the viral binding site , particularly the arginine side chains , is adaptable to different glycan structures .
the prior aav2/heparin structures differed in the exact location of perceived heparin density and the need ( or not ) for conformational changes that would allow interactions with genetically implicated arginines.10 , 39 in the study at 18   resolution , 1  difference peaks , interpreted as heparin , required a hypothesized glycan - induced protein conformational change for interaction with the arginines .
difference density at 0.5  near the tips of the 3-fold spikes and near the hi  loop was cited in support of a postulated heparin - triggered allosteric change that would widen a channel along the 5-fold axis for subsequent dna release .
even though the fondaparinux might have partial occupancy in the current study , its density is 10-fold stronger and more robust statistically .
differences with the 18   heparin study reflect not only improved resolution , but , we believe , the on - grid preparation of the complex used here and in the 8   heparin study .
this minimizes aggregation and increases the fraction of em - observable particles that are in the bound state .
the fondaparinux location is consistent with the 8   heparin and 5   sos studies,8 , 9 contacting directly the genetically implicated arginines without the conformational changes proposed in the 18   study .
the current 2.8   resolution should be sufficient for such conformational changes to be clear .
however , no evidence is seen of a substantial shift of the refined model , or of paired positive and negative difference density peaks that would be expected .
subtle , rather than large , conformational change is reminiscent of the crystal structures of foot - and - mouth disease viruses ( fmdvs ) complexed with heparinoid oligosaccharides.45 , 46 , 47 so too are pre - formed sulfate - binding sites containing arginines , and natural or in  vitro modulation of binding affinity and infectivity upon mutation of these residues.13 , 46 the findings with aav and fmdv are all in contrast with hiv with its cd4-induced changes to gp120 that modulate binding to heparan sulfate , and the structural changes in heparan sulfate binding domains that are key to hiv co - receptor interactions .
thus , among viral proteins , a wide spectrum of glycan interactions is emerging , from rigid to flexible .
it remains to be seen whether this proves to be more common among non - enveloped viruses with no need for attachment to trigger membrane fusion .
the absence of widespread conformational changes in aav is consistent with recently changed perception of the role of glycan - binding during infection .
hspg was originally thought to be a primary cell receptor with various membrane proteins suggested to have secondary roles during endocytic entry .
recently , the gene product kiaa0319l ( also known as aavr ) has been identified as a protein essential for aav entry , with many of the properties classically associated with viral receptors .
furthermore , at high multiplicity of infection , overexpression of kiaa0319l can overcome the inhibitory effect of a heparan synthesis cell knockout .
the results of this research add to the emerging picture that the glycan binding site can tolerate lower specificity interactions than once anticipated .
glycan binding might serve a more accessory role in adherence , so that the virus is anchored in close proximity to the cell surface , thereby maximizing the chance of binding to a receptor that is transiently exposed on the surface .
empty capsids of aav - dj were expressed in sf9 insect cells using the  bac - to - bac baculovirus expression vector system ( invitrogen ) as previously described , except that aav - dj substituted for aav-2.12 , 50 the vlps were purified , as before , using three rounds of cscl density gradient ultracentrifugation , followed by heparin affinity chromatography , eluting with a nacl gradient .
capsids were then diluted in 50  mm hepes , 25  mm mgcl2 , 25  mm nacl ( ph 7.4 ) , or , for spr , into 137  mm nacl , 2.7  mm kcl , 10  mm na2hpo4 , and 1.8  mm kh2po4 ( ph 7.4 ) .
fondaparinux ( arixtra ; molecular weight [ mw ]  = 1,728 ) was from glaxosmithkline and mylan .
heparin oligosaccharides , classified by dp , included tetrasaccharide ( dp4 ) and hexasaccharide ( dp6 ) , prepared by partial hydrolysis using heparin lyase 1 of bovine lung heparin ( sigma ) followed by size fractionation .
spr measurements were performed on a biacore 3000 and analyzed with the biaevaluation software ( version 4.0.1 ) .
biotinylated heparin was prepared by reaction of sulfo - n - hydroxysuccinimide long - chain biotin ( thermo scientific ) with unsubstituted glucosamine amino groups in the polysaccharide chain and was immobilized to a streptavidin ( sa ) chip based on the manufacturer s protocol . in brief , 20  l of the heparin - biotin conjugate ( 0.1  mg / ml ) in hbs - ep running buffer ( ge healthcare ) was injected over flow cell 2 ( fc2 ) of the sa chip , at 10  l / min .
immobilization was confirmed by an 50 ru increase in the sensor chip . the control flow cell ( fc1 )
aav at 8.3 pm ( equivalent to capsid protein at 0.5  nm ) was diluted in hbs - ep buffer ( 0.01  m hepes , 0.15  m nacl , 3  mm edta , 0.005% surfactant p20 [ ph 7.4 ] ) and injected at 30  l / min . the same buffer was flowed through , during 3  min of dissociation . the sensor surface was regenerated with 30  l of 0.5% sds , then with 2  m nacl .
inhibition was measured by pre - mixing the aav sample with fondaparinux at 120  m , or , for comparison , tetrasaccharide ( dp4 ) , hexasaccharide ( dp6 ) at 1  m , or sos or heparin at 0.1  m , and the binding to heparin was re - measured as above . after each competition assay , aav binding was re - measured to ensure that the surface had been completely regenerated .
hela s3 cells ( atcc ccl-2.2 ) were plated at 1.1   10 cells / well in 96-well plates ( falcon , becton dickinson ) and cultured overnight in f-12k medium ( hyclone , ge life sciences ) , supplemented with 10% fetal bovine serum .
the specified concentrations of fondaparinux were introduced to the medium 1015  min before cells were transduced with the self - complementary vector scaav2-cmv - mcherry ( university of north carolina vector core ) at an moi of 1.2   10 viral genomes per cell ( vg / cell ) .
cells were incubated for 25  hr at 37c , at which time they were trypsinized and assayed for transgene expression by flow cytometry tuned to pe / cy-5 on a macsquant analyzer 10 ( miltenyi biotec ) .
aggregation was observable when mixed in bulk solution , presumably because of oligosaccharide cross - linking polyvalent aav particles .
aav - dj ( 3  l ) was applied to copper grids that had been glow discharged in 75%/25% ar / o at a concentration of 0.6  mg / ml . for complex , the grid was hand blotted before adding 3  l 5.7  mm fondaparinux in ultrapure water for 15 s , giving a > 600-fold excess of ligand over virus subunits .
all grids were vitrified in liquid nitrogen - cooled ethane using an fei vitrobot ( fei ) with a single 3  s blot of force 1 at 100% humidity and 4c .
images were acquired using an fei titan krios ( fei ) using leginon with a total dose of 52.63 e /  .
the images were recorded on a de-20 direct electron detector ( direct electron ) , dose fractionated across 18 frames with a dose rate of 2.9 e /  per frame .
defocus was randomly set between 2.0 and 3.5  m , with defocus estimates made continually throughout image acquisition using the automated contrast transfer function ( ctf ) estimator ace and ctffind3 .
for the aav - dj / fondaparinux complex , two datasets were obtained from the same grid at a magnification of 29,000 , but with differing defocus ranges .
images were acquired using an fei titan krios ( fei ) using leginon with a total dose of 66 e /  , fractionated across 45  frames at 1.5 e /  per frame .
the first dataset was collected with defocus between 1.5 and 3.0  m , whereas the second was between 0.75 and 1.75  m ( using defocus estimates as above ) .
frame alignment and dose compensation were performed with version 2.5.1 of software provided by direct electron . in brief , rolling frame averages are iteratively aligned to the current sum of frames .
dose compensation is through gaussian low - pass filtering to a frame resolution that is dependent on the cumulative dose , using resolutions calibrated from attenuation of the electron diffraction of catalase crystals ( direct electron ) . following alignment and dose compensation for entire frames ,
the first datasets for the aav / fondaparinux complex yielded 503 images and 65,978 particles , and the second yielded 548 images and 54,188 particles .
particle picking was semi - automatic using findem within appion and using a template that was a rotational average of manually picked particles . particles over carbon
initial orientations were generated using eman1 by refining against the structure of aav , low - pass - filtered to 20   resolution , with two iterations and an angular increment of 1. the euler angles were then further refined with nine iterations of frealign , constraining 60-fold icosahedral symmetry throughout image processing .
it was expected to be challenging , because the ligand constitutes only 2.5% of the mass .
indeed , the reconstruction for the class with higher occupancy was only marginally different , so the full dataset was used thereafter .
reconstructions were sharpened to correct for high - frequency attenuation using em - b factor with default parameters .
envelope corrections , together with the effective em magnifications and resolutions , were least - squares refined , using rsref , to maximize agreement between the prior 4.5   aav - dj atomic structure and the new 2.8 and 3.5   maps .
( resolution is refined by adjusting the limit on a butterworth low - pass filter applied to the atomic model , until its density optimally agrees with the experimental reconstruction , thereby cross - checking the fsc - derived estimates . ) through the calibration to the atomic model , reconstructions of the fondaparinux complex and uncomplexed aav - dj were put on uniform scales  in map values and in magnification .
structure factors for the 3.5   native were scaled in reciprocal space to the 2.8   reconstruction of the complex by resolution shell using eman , after low - pass filtering both reconstructions to 6 , 5 , or 4   resolution .
the 5   difference map was used to model the fondaparinux , starting with the structure from pdb : 4x7r . because the occupancy of the glycan site was less than unity , density for the viral protein in the complex represented a mixture of bound and unbound conformations .
the bound state was revealed through a difference map weighted by the ligand occupancy , o:=complex[1ofondaparinux].aav an initial estimate for the occupancy came by adjusting o until density for the glycan and viral protein were commensurate .
after modeling the glycan and the glycan - bound configuration of the hbd loop , the estimate of the occupancy was refined using rsref such that the weighted sum of bound and unbound atomic models gave best agreement with the observed reconstruction of the complex .
the process was repeated through iterations of atomic refinement , because an improved model allowed an improved estimate of the occupancy and hence improvement in the weighted difference map .
structures for the viral protein were refined by simulated annealing torsion angle optimization into the native and weighted difference reconstructions .
refinement used a real - space objective function that calculates the density of each map grid point from the contributions of neighboring atoms , accounting for the effects of resolution and the em envelope .
this objective function was embedded in the crystallographic refinement program cns v1.3 to take advantage of its stereochemical restraints and a low - parameter torsion angle optimizer that mitigates over - fitting .
an additional restraint was added through a flat - bottom potential to ensure that  and  backbone dihedrals did not deviate from the allowed areas of a ramachandran plot .
stereochemical restraints for the fondaparinux were based on the nto entry in the library of the ccp4 suite , with the addition of restraints toward preferred glycosidic torsion angles .
pucker of the pyranose rings was checked post facto through calculation of cremer - pople parameters , but not enforced as an explicit restraint68 , 69 ( http://www.ric.hi-ho.ne.jp/asfushi/ ) .
; visualization , q.x . ; supervision , s.m.s . , r.j.l . , and m.s.c .
 atomic structures of adeno - associated virus ( aav)-dj , alone and in complex with fondaparinux , have been determined by cryoelectron microscopy at 3   resolution . 
 the gene therapy vector , aav - dj , is a hybrid of natural serotypes that was previously derived by directed evolution , selecting for hepatocyte entry and resistance to neutralization by human serum . 
 the structure of aav - dj differs from that of parental serotypes in two regions where neutralizing antibodies bind , so immune escape appears to have been the primary driver of aav - dj s directed evolution . 
 fondaparinux is an analog of cell surface heparan sulfate to which several aavs bind during entry . 
 fondaparinux interacts with viral arginines at a known heparin binding site , without the large conformational changes whose presence was controversial in low - resolution imaging of aav2-heparin complexes . 
 the glycan density suggests multi - modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer , consistent with a role in attachment , prior to more specific interactions with a receptor protein mediating entry . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest