in recent years , there 
 is increasing evidence to suggest that prokaryotes 
 adopt primitive organelle - like structures called bacterial microcompartments 
 or nanocompartments , depending on their size .
such assemblies localize 
 and compartmentalize multiple enzymes and substrates involved in specific 
 metabolic pathways . the ability to mimic 
 and understand enzymatic activity in confinement would provide ground - breaking 
 insight into these assemblies and in organelles in general .
one of the main challenges is to controllably 
 package and coencapsulate 
 different enzymes noncovalently within the same compartment as exemplified 
 by nature .
there has been some success using coiled - coil helices , peptide tags , and 
 protein  protein fusion constructs to direct enzymatic cargo encapsulation into protein cages . however , 
 such approaches often lead to covalently connected protein cargo or 
 inefficient loading ( i.e. , formation of empty cage assemblies ) . to 
 circumvent these problems ,
we seek a versatile approach that would 
 promote a noncovalent co - encapsulation of enzymes within a single 
 protein cage in vitro .
the cowpea chlorotic mottle virus ( ccmv ) 
 is an ideal candidate 
 to mimic bacterial nanocompartments , owing to its size and biocompatibility . 

the ccmv capsid is 28 nm in diameter and is based on a t = 3 lattice ( t , triangulation number ) , with 12 
 pentamers and 20 hexamers of identical monomers of capsid protein 
 ( cp ) organized as 90 dimers .
similar to 
 bacterial compartments , it has multiple 
 pores in the capsid shell ( around 2 nm ) , which allows molecules and substrates to diffuse in and out .
the 
 cp n - terminal region is enriched in positively charged residues , termed 
 the arginine - rich motif ( arm ) , that face the capsid inner surface . 

after removal of native single - strand ( ss ) rna cargo , the arm can 
 trigger reassembly of capsid protein dimers in the presence of an 
 appropriate negatively charged template , resulting in the formation 
 of monodisperse virus - like particles ( vlp ) .
encapsulation 
 of enzymes in vlps has been shown to stabilize and protect the enzymes , 
 and its ease of modification enables new applications . in this contribution ,
single- and complementary - stranded 
 dna tags 
 are chemically attached to the exterior of chosen enzymes , resulting 
 in negatively charged complexes that induce the co - encapsulation inside 
 ccmv capsids . unlike covalent interactions , electrostatic interactions 
 between the dna tags and the interior of the capsid
provide a tunable 
 system , enabled , for instance , by changing the salt concentration 
 or by varying the length of the dna chains .
hence , this makes our 
 system a model for natural bacterial compartments ( e.g. , the encapsulins ) 
 where the confined enzymes are not covalently bound but rather included 
 in the protein cage by noncovalent , multivalent interactions .
noncovalent encapsulation mediated by nucleic 
 acid tags has been reported with the use of genetic engineering and/or only focused on a single enzyme . using this strategy , we were able to confine 
 two separate cascade systems in vitro , for which the glucose oxidase 
 ( gox ) , a 160 kda dimeric enzyme , is chosen as the primary enzyme for 
 both encapsulated cascades .
gox catalyzes 
 the oxidation of glucose into gluconolactone ( which undergoes spontaneous 
 hydrolysis into gluconic acid ) and produces 
 hydrogen peroxide as the side product . in the first cascade system , hydrogen peroxide produced by 
 gox 

is consumed by the so - called dnazyme , a peroxidase - mimic formed in 
 situ by a specific sequence of ssdna in the presence of hemin ( figure 1a ) . in the second cascade system , in the presence of atp and 
 nadp , gluconic acid produced by gox
is consumed by a secondary 
 enzyme , gluconokinase ( gck ) that is coencapsulated inside the ccmv - like 
 particles , followed by a nonencapsulated tertiary enzyme , 6-phosphogluconate 
 dehydrogenase ( 6-pgdh ) , to form ribulose-5-phosphate and nadph , the latter of which can be monitored spectroscopically 
 ( figure 1b )
. therefore , 
 the nucleic acid tags in this work are useful both as a secondary 
 biocatalyst ( in cascade system i ) and as negatively charged tags to 
 trigger the encapsulation of the enzyme(s ) ( in cascade systems i and 
 ii ) .
schematic representation of the enzyme pathways ( encapsulated processes 
 shown in gray boxes ) .
gox oxidizes glucose to gluconic acid and produces h2o2 , which dnazyme uses for subsequent reaction with abts inside 
 ccmv capsid .
the conversion of glucose to d - gluconate-6-p occurs 
 at the interior of the ccmv capsids , whereas the conversion of d - gluconate-6-p into ribulose-5-p occurs at the exterior of 
 the ccmv capsid catalyzed by tertiary enzyme , 6-pgdh .
we 
 anticipated that functionalization and subsequent hybridization of 
 gox and gck with ( complementary ) single - stranded dna strands should 
 promote their co - encapsulation into ccmv capsids ( figure 2 ) .
we suspect that the size 
 of the enzyme cargo as well as the number and spatial distribution 
 of negative charges anchored to the enzyme surface might play a role 
 in determining the efficiency of the encapsulation . to modify and 
 hybridize the relevant enzymes , the lysine residues of gox and gck 
 were functionalized with a heterobifunctional linker using sulfo - nhs 
 coupling followed by maleimide 
thiol chemistry on the single 
 stranded dna ( ssdna ) or its complementary sequence ( csdna ) , respectively 
 ( see figures s1 and s2 ) . all hybridized 
 complexes were purified initially by spin - filtration to remove excess 
 dna .
furthermore , size - exclusion chromatography ( sec ) was used for 
 the gox  gck dual - enzyme complex to remove nonhybridized gck 
 and gox prior to encapsulation ( figure s3 ) .
encapsulation of different enzyme  dna hybrids inside ccmv 
 capsids ( gray ) at ph 7.5 .
encapsulation of ( a ) ssdna in yellow , ( b ) 
 gox , in blue , functionalized with ssdna , ( c ) gck , in green , functionalized 
 with the complementary ssdna in red , ( d ) gox conjugated to gck .
the 
 specific sequence of ssdna is catalytically active in the presence 
 of hemin .
encapsulation of gox  ssdna , 
 gck  csdna , or hybridized 
 gox  gck in ccmv at ph 7.5 led to the formation of stable capsid - like 
 assemblies , which were purified by sec ( figure 3a  d ) .
the elution volume ( 12 ml ) , 
 together with the relative absorbance ratio ( 260 nm/280 nm ) > 1 , 
 are 
 characteristic features of intact ccmv capsids containing dna - based 
 cargo .
control experiments with no dna tags ( i.e. , nonfunctionalized 
 gox and/or gck ) confirmed that enzymes lacking ssdna or csdna can not 
 be encapsulated ( figure s4 ) .
as anticipated , 
 the dna strands provide the required negative charges for reassembly 
 into virus - like particles .
this further ensures that no empty particles 
 are obtained with this strategy and that formed particles always contain 
 a negatively charged cargo .
size - exclusion chromatograms for ccmv containing ( a ) ssdna , ( b ) gox  ssdna , 
 ( c ) gck  csdna , and ( d ) gox  gck , with monitoring at  
 = 260 ( red ) , 280 ( blue ) , and 450 nm ( black ) , for dna , ccmv , and flavin 
 ( gox ) , respectively .
negatively stained transmission electron 
 microscopy of ( e ) ssdna , ( f ) gox  ssdna , ( g ) gck  csdna , 
 and ( h ) gox  gck encapsulated ccmv assemblies .
the assembled ccmv - like particles were characterized 
 by negative 
 staining transmission electron microscopy ( tem ) ( figure 3e  h ) and dynamic light scattering ( dls , figure s5 ) , showing spherical structures of around 20 nm in 
 diameter .
the size of around 20 nm indicates the formation of t = 1 icosahedral symmetry that is composed of 60 identical 
 capsid subunits .
furthermore , co - encapsulation 
 of gox and gck in a single particle was confirmed with sds - page and 
 western blot analyses ( figures s6 and s7 ) .
additionally , their concentrations and relative ratios were estimated 
 by gel densitometry , which suggested a gox / gck / capsid protein ratio 
 of approximately 1:1.4:60 . since the capsid protein
is composed of 
 60 identical subunits , we estimate that only a single gox 
ssdna 
 is confined inside ccmv - like particles for cascade system i and a 
 hybrid of 1 gox  dna and 1 or 2 gck 
csdna is confined 
 for cascade system ii . to 
 confirm the assembly of ccmv - like particles with t = 1 icosahedral symmetry
, we analyzed the gox  ssdna - loaded 
 ccmv - like particles with cryo - electron microscopy ( cryo - em ) to calculate 
 their native three - dimensional reconstruction ( 3dr ) ( figure 4a ) .
the sample contained particles 
 with spherical and elongated profiles as well as irregular assemblies 
 ( figure 4a , inset ) . 

two - dimensional classification followed by a three - dimensional classification 
 using relion software resulted in two sizes of icosahedral capsids 
 with t = 1 architecture . whereas class i capsids were 214  in diameter ( figure 4b ) , class ii capsids were 226 
  ( figure 4c ) .
three - dimensional 
 cryo - em reconstructions of gox  ssdna - loaded 
 ccmv capsids .
( a ) cryo - electron micrograph of gox  ssdna - loaded 
 ccmv capsids .
two - dimensional class averages derived 
 from the final 15481 particle data set ( inset ) .
( b ) surface - shaded 
 representation of the outer surface of the class i t = 1 capsid ( diameter 21.4 nm ) viewed along a 2- , 3- , and 5-fold 
 axis of icosahedral symmetry ( top to bottom ) .
models of the class 
 i t = 1 capsid , with the front half of the cargo 
 and protein shell removed ( right ) .
( c ) 
 surface - shaded representations of the outer surface of the class ii t = 1 capsid ( diameter 22.6 nm ) ( as in b ) .
capsids i and ii made up 50% of the total particles 
 in the sample 
 ( 70% class i , 30% class ii ) .
the cryo - em images analyzed for 
 processing are in fact snapshots of the dynamic states of the sample ; 
 the ratio observed could be due to displacement of dynamic equilibrium 
 toward class i t = 1 capsids ( 70% class i , 30% class 
 ii ) .
the two particle sizes might be related to the reported dynamic 
 swelling of the t = 3 ccmv native capsid , as the 
 size difference of 5% involves a 7  outward radial expansion 
 and widening of the pores , hinting at possible structural breathing . 
 in both t = 1 capsids ,
the pentamer bases were strongly 
 connected to the underlying gox  ssdna cargo , although the capsid 
 surface pores were distinct . whereas class i capsid pentamers barely 
 left any space between their lateral contacts , those of class ii capsids 
 were clearly separated and left large pores , especially at the icosahedral 
 2-fold axes ( figure 4b , c , arrows ) .
docking of ccmv capsid protein ( cp ) dimer into 
 the cryo - em density 
 maps of gox - ssdna - loaded t = 1 vlp showed major structural differences 
 of the two classes ( figure 5a , b ) .
connecting densities between pentamers and cargo were 
 mediated by residues 4250 of the cp n - terminal region ( figure 5c , dark blue ) , although 
 the preceding region ( residues 2741 ) could also be involved 
 ( figure 5c , pink ) . 

the cp c - terminal ends were responsible for cp dimer assembly in class 
 i ccmv t = 1 capsids ( figure 5a , arrows ; figure 5d , red ) . the hinge angle formed between cp 
 dimers in gox 
ssdna - loaded t = 1 class i capsids 
 was 60 ( figure 5d ) ; it resembles that found at the quasi-2-fold axes of the 
 swollen t = 3 ccmv capsid and in other cp dimers such as the phthalocyanine - loaded t = 1 vlp .
ccmv cp dimers are 
 the building blocks of native t = 3 virion capsids 
 as well as of in vitro assembled structures such as tubes and icosahedral capsids with t = 1 ( containing 30 cp dimers ) , t = 2 ( 60 dimers ) , 
 and t = 3 ( 90 dimers ) architecture .
the outward expansion of the class ii capsid pentamer entailed 
 the disappearance of or a great reduction in dimeric contacts ( figure 5b , arrows ) and indicated 
 that these interactions contribute much less to class ii capsid stability 
 than to that of class i capsids .
assuming the same building block 
 is involved , the class ii capsids are based on pentamers bound weakly 
 by the cp c - terminal ends ( figure 5e , red ) , which adhere strongly to the polyanionic cargo .
pseudoatomic 
 model of gox  ssdna - loaded ccmv capsids . ( a ) t = 1 class i capsid viewed down a 3-fold axis from outside , 
 with docked ccmv cp atomic coordinates .
( b ) class ii t = 1 capsid viewed down a 3-fold axis from outside ( as 
 in b ) .
( c ) pentamer contacts with the cargo mediated by residues 4250 
 in the n - terminal region ( dark blue , bottom view ) . the n - terminal 
 region residues 2741 might also contribute to cargo 
side view ( top ) , top view ( bottom ) . the 
 hinge dihedral angle is indicated .
cp monomers in the class ii dimer 
 are 6.5  further apart than class i dimers ( n - terminal 2732 
 region is omitted ) . after imposing icosahedral 
 symmetry in class i and ii capsids
, 
 we observed the packed cargo as a hollow sphere ( 9.6  10 and 1.2  10  , respectively ) , 
 with numerous connections to the t = 1 capsid inner 
 surface .
based on the atomic model of gox ( pdb 1gal ) , several copies 
 of gox could be encapsulated in the capsid , although our biochemical 
 analyses indicated the presence of a gox dimer only .
this discrepancy 
 is probably due to the chemical modification of accessible lys residues 
 of gox that are covalently bound to ssdna .
capsid 
 connections observed in the 3d cryo - em maps probably represent the 
 interaction of the arm region with negatively charged dna strands . 

gox  ssdna packaging resulted in a slightly disordered icosahedral 
 capsid ( also reflected in a limited map resolution ) , but this cargo 
 enabled structural polymorphism with weak cp interactions in the dimer . 

both t = 1 capsids coexist in dynamic equilibrium , 
 probably enabled because the cp  ssdna interactions are more 
 flexible ( or less well - defined ) than the cp  ssrna interactions . 
 to our knowledge , this is the first demonstration of the formation 
 of a t = 1 ccmv - like structure templated by a biological 
 soft material that also displays an extreme capsid swelling . following 
 structural characterization of enzyme  dna complexes inside t = 1 ccmv - like particles
, we proceeded to monitor the enzymatic 
 activity of both cascade systems to examine whether the encapsulated 
 complexes were still catalytically active . for cascade system
i ( figure 1a and figure 6a ) , we deliberately chose the 
 ssdna sequence coupled to the gox to be that of a hemin - binding dna 
 quadruplex , the so - called dnazyme .
in the presence of hemin , the ssdna 
 spontaneously forms a scaffold that mimics the catalytic properties 
 of horseradish peroxidase ( hrp ) .
dnazyme was monitored via the production 
 of abts at  = 410 nm upon addition of glucose 
 to the system .
the activity plot obtained 
 for encapsulated gox  dnazyme shows that both gox and dnazyme 
 remained catalytically active after encapsulation ( figure 6b and figure s8a ) .
an increase in both km values ( 2.2-fold ) and kcat values ( 1.7-fold ) 
 is observed when the system is encapsulated ( summarized in table s1 ) .
( a ) schematic representation 
 of cascade system i in the presence of a competing enzyme , catalase 
 ( encapsulated processes shown in gray boxes ) .
( b ) kinetic measurements 
 of cascade system i ; the production of abts was 
 monitored at  = 410 nm at different glucose concentrations . 

( c ) kinetic measurements of cascade system ii ; the production of nadph 
 was monitored at  = 340 nm at different glucose concentrations . 

( d ) kinetic measurements for the production of abts in the presence of and after ph inactivation of the competing enzyme , 
 catalase . for cascade system
ii consisting 
 of gox , gck , and 6-pgdh ( figure 1b ) , we monitored 
 the formation of the end product , nadph at  = 340 nm , upon 
 addition of glucose to the system . both 
 enzymes ( gox and gck )
are therefore required for the reaction and 
 its visualization at 340 nm . the activity profile in figure 6c and
figure s8b confirms that both enzymes were present in the system and 
 still active upon hybridization and subsequent encapsulation .
while the km values remain similar 
 for both systems , the kcat values show 
 a 2-fold increase for the encapsulated system ( summarized in table s1 ) .
based on the recurring trends , 
 a slightly higher turnover number 
 ( kcat ) upon pathway encapsulation is estimated , 
 although the protein concentration determination by gel densitometry 
 is expected to have a large deviation and consequently also the kcat . an eventual increase might be the result 
 of a local enhancement in effective molarity due to confinement or of the channeling effect when multiple enzymes 
 in a cascade pathway are brought to a close proximity inside a confined 
 system .
the enzymatic activities 
 observed for both cascade pathways indicated 
 that the substrate glucose was able to diffuse into the capsid shell . 

we further investigated whether the intermediate of the cascade could 
 also diffuse freely or was trapped inside the cagelike structure during 
 the reaction . in order to confirm the state of the intermediate
, h2o2 is broken down to water and oxygen 
 by the enzyme catalase , and it can therefore act as an external competitor 
 with the dnazyme .
in contrast , if the 
 h2o2 intermediate is trapped inside ccmv ( as 
 proposed for the bacterial microcompartments ) , the kinetics of abts production should 
 remain unaltered .
instead , we observed almost complete suppression 
 of abts production in the presence of catalase 
 ( figure 6d ) . only upon 
 lowering the buffer to ph 4 and hence inactivating catalase ( t = 60 min )
could the h2o2 conversion 
 by dnazyme be restored while maintaining the stability of the particles , 
 as confirmed by sec and tem analyses in figure s9 .
taken together , in agreement 
 with a previous report , we also observed 
 that the ccmv capsid shell is permeable to small molecules such as 
 h2o2 , which can diffuse out of the capsid shell 
 and react with the competing enzyme .
nevertheless , it should be noted 
 that the dnazyme is likely to exhibit lower catalytic efficiency and 
 lower affinity to h2o2 compared to catalase , which could also lead to the diffusion of h2o2 out of the ccmv capsid .
we have presented a highly effective strategy of using single - stranded 
 dna for the controlled noncovalent packing of enzyme cascades in a 
 single protein capsid assembly . to demonstrate the versatility of 
 this strategy , two different cascade systems based on glucose oxidase 
 were assembled inside the protein shell of ccmv at ph 7.5 and
this encapsulation strategy 
 resulted in icosahedral structures of approximately 20 nm , which were 
 further analyzed with 3d cryo - em .
the resulting 3d reconstruction 
 provides the first - time demonstration of t = 1 structured 
 assemblies of ccmv around a biological soft matter template .
in addition , 
 an extra - swelling phenomenon was indicated on the basis of the coexistence 
 of two differently sized particles of similar structure and origin . 

the method presented for assembling virus - like particles can provide 
 a structural and functional basis to analyze bacterial protein organelles 
 and will further improve our understanding of their containment properties 
 and biochemical function .
the ssdna ( 5-hs-(ch2)6-gggtagggcgggttgggtttt-3 ) 
 and csdna ( 5-hs-(ch2)6-aaaacccaacccgccctaccc-3 ) 
 oligonucleotide sequences were synthesized by eurofins mwg operon . 
 for the coupling of dna to enzymes ,
the bifunctional cross - linker 
 sulfo - emcs ( n-[-maleimidocaproyloxy ] sulfosuccinimide 
 ester ) was purchased from pierce .
d - gluconate / d - glucono--lactone 
 assay kit was purchased from megazyme and used as provided .
all other 
 reagents were purchased from sigma - aldrich or fluka unless stated 
 otherwise and were used without further purification .
a stock solution 
 of hemin ( 5 mm ) was prepared in dmso and stored in the dark at 4 c . 

hcl , 500 mm nacl , 
 50 mm mgcl2 , 1 mm dtt , ph 7.5 ) to obtain ccmv dimer coat 
 proteins ( ccmv - cp ) ( 500 m ) .
gck , 
 and gox  gck were buffer exchanged against milli - q water using 
 amicon ultra centrifugal filters ( 30 kda or 10 kda mwco ) .
gck 
 and ccmv  cp ( in assembly buffer ) were mixed in a 4:1 ( v / v ) 
 ratio and incubated for 2 h at 4 c before purification by size - exclusion 
 chromatography ( sec ) using a superose 6 10/100 gl column , eluting 
 with 50 mm tris 
hcl , 100 mm nacl , 10 mm mgcl2 , 0.5 
 mm dtt at ph 7.5 .
protein fractions were collected and analyzed by 
 sds  page , agarose gel , and western blot analysis . for the complete 
 procedure ,
samples ( 5 
 l ) were applied onto formvar  carbon - coated grids .
uranyl acetate ( 5 l , 
 1% w / v ) was added and the excess liquid was drained after 20 s and 
 dried for 30 min at room temperature .
the samples were examined on 
 a feg - tem ( phillips cm 30 ) operated at 300 kv acceleration voltages .
ssdna - loaded 
 vlp ( 5 l ) were applied to one side of quantifoil r 2/2 holey 
 grids , blotted , and plunged into liquid ethane in a leica em cpc cryofixation 
 unit .
the grids were analyzed in a tecnai g2 electron microscope equipped 
 with a field emission gun operating at 200 kv , and images were recorded 
 under low - dose conditions with a fei eagle ccd at a detector magnification 
 of 69,444x ( 2.16  / pixel sampling rate ) .
image processing operations 
 were performed using xmipp and relion , and graphic representations were produced with 
 ucsf chimera .
the xmipp automatic picking 
 routine was used to select 15481 particles , and defocus was determined 
 with ctffind .
images were 2d - classified 
 using the appropriate relion routine and 7932 isometric particles 
 were selected .
the structure of phthalocyanine - loaded ccmv t = 1 capsid was filtered out to 30  , and the cargo 
 density was masked .
this map was used as an initial model for 3d classification 
 of spherical particles , using relion to select 5572 ( class i ) and 
 2318 ( class ii ) particles ; these data sets were used to obtain the 
 final 3drs using the relion autorefinement routine .
resolution was 
 assessed by gold standard fsc between two independently processed 
 half - data sets . applying a correlation limit of 0.5 ( 0.3 ) ,
the resolution 
 for class i and ii 3d maps was 22.7 ( 22.2 ) and 25.6 ( 21.3 )  , 
 respectively .
the chimera fitting tool was used to dock the atomic 
 structure of a whole pentamer from the x - ray structure of ccmv ( pdb entry 1cwp ) into the cryoem maps .
substrate solutions containing 
 various glucose concentrations ( 01 
 m , 180 l ) were prepared .
an enzyme solution containing either 
 ( 1 ) free gox  gck ( 60 l ) or ( 2 ) encapsulated gox 
6-pgdh ( 55 u / ml , 2 l ) 
 and 16.1 mm nadp+ containing 69.4 mm atp were added to each reaction 
 mixture at ph 7.5 , according to the manufacturer s instructions 
 ( megazyme kit ) .
the reaction was started upon addition of glucose 
 ( 120 l ) to enzyme ( 82 l ) , and formation of reduced nadph 
 was monitored at  = 340 nm in 100 s time intervals over 2 h 
 at 27 c .
stock solutions containing both substrates glucose 
 ( ranging from 
 01 m ) and 4 mm abts were freshly prepared at room temperature . 

dnazyme 
 ( 30 l ) and hemin ( 30 l , 5 m ) or ( 2 ) encapsulated 
 gox  dnazyme ( 30 l ) and hemin ( 30 l , 5 m ) 
 were prepared and incubated at rt for 2 h. to each enzyme containing 
 solution ( 60 l ) was added the substrate solution containing 
 both glucose and abts ( 120 l ) , and the reaction was monitored 
 immediately at  = 410 nm for the conversion of abts to abts at 27 c in 100 s time intervals over 2 h. control 
 experiments containing hemin , glucose , and abts were performed under 
 the same reaction conditions .
experimental data were corrected for 
 background absorbance ( using the control experiment as a reference ) . 
 the concentration of abts or nadph was determined 
 using the lambert 
beer law , assuming extinction coefficients 
 of abts ( 410  nm = 36000 
 m cm ) or nadph ( 340  nm = 6300 m cm ) before plotting concentration ( m ) vs time ( min ) curves , 
 from which the velocity ( v ) was determined ( m / min ) . a dilution 
 factor ( df ) relative to the enzyme ( gox ) and a proportionality factor 
 of abts to substrate consumption ( p = 1/2 ) were used 
 to correct the velocity values as described in eq 1.1
 the 
 packaging of proteins into discrete compartments is an essential 
 feature for cellular efficiency . 
 inspired by nature , we harness virus - like 
 assemblies as artificial nanocompartments for enzyme - catalyzed cascade 
 reactions . using the negative charges of nucleic acid tags , we develop 
 a versatile strategy to promote an efficient noncovalent co - encapsulation 
 of enzymes within a single protein cage of cowpea chlorotic mottle 
 virus ( ccmv ) at neutral ph . the encapsulation results in stable 2122 
 nm sized ccmv - like particles , which is characteristic of an icosahedral t = 1 symmetry . 
 cryo - em reconstruction was used to demonstrate 
 the structure of t = 1 assemblies templated by biological 
 soft materials as well as the extra - swelling capacity of these t = 1 capsids . 
 furthermore , the specific sequence of the 
 dna tag is capable of operating as a secondary biocatalyst as well 
 as bridging two enzymes for co - encapsulation in a single capsid while 
 maintaining their enzymatic activity . 
 using ccmv - like particles to 
 mimic nanocompartments can provide valuable insight on the role of 
 biological compartments in enhancing metabolic efficiency . 