Structural	B-experimental_method
characterization	I-experimental_method
of	O
encapsulated	B-protein_state
ferritin	B-protein_type
provides	O
insight	O
into	O
iron	B-chemical
storage	O
in	O
bacterial	B-taxonomy_domain
nanocompartments	B-complex_assembly

Ferritins	B-protein_type
are	O
ubiquitous	O
proteins	O
that	O
oxidise	O
and	O
store	O
iron	B-chemical
within	O
a	O
protein	O
shell	B-structure_element
to	O
protect	O
cells	O
from	O
oxidative	O
damage	O
.	O

We	O
have	O
characterized	O
the	O
structure	B-evidence
and	O
function	O
of	O
a	O
new	O
member	O
of	O
the	O
ferritin	B-protein_type
superfamily	O
that	O
is	O
sequestered	O
within	O
an	O
encapsulin	B-protein
capsid	O
.	O

We	O
show	O
that	O
this	O
encapsulated	B-protein_state
ferritin	B-protein_type
(	O
EncFtn	B-protein
)	O
has	O
two	O
main	B-structure_element
alpha	I-structure_element
helices	I-structure_element
,	O
which	O
assemble	O
in	O
a	O
metal	B-protein_state
dependent	I-protein_state
manner	O
to	O
form	O
a	O
ferroxidase	B-site
center	I-site
at	O
a	O
dimer	B-site
interface	I-site
.	O

EncFtn	B-protein
adopts	O
an	O
open	B-protein_state
decameric	B-oligomeric_state
structure	B-evidence
that	O
is	O
topologically	O
distinct	O
from	O
other	O
ferritins	B-protein_type
.	O

While	O
EncFtn	B-protein
acts	O
as	O
a	O
ferroxidase	B-protein_type
,	O
it	O
cannot	O
mineralize	O
iron	B-chemical
.	O

Conversely	O
,	O
the	O
encapsulin	B-protein
shell	B-structure_element
associates	O
with	O
iron	B-chemical
,	O
but	O
is	O
not	B-protein_state
enzymatically	I-protein_state
active	I-protein_state
,	O
and	O
we	O
demonstrate	O
that	O
EncFtn	B-protein
must	O
be	O
housed	O
within	O
the	O
encapsulin	B-protein
for	O
iron	B-chemical
storage	O
.	O

This	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
is	O
widely	O
distributed	O
in	O
bacteria	B-taxonomy_domain
and	O
archaea	B-taxonomy_domain
and	O
represents	O
a	O
distinct	O
class	O
of	O
iron	B-chemical
storage	O
system	O
,	O
where	O
the	O
oxidation	O
and	O
mineralization	O
of	O
iron	B-chemical
are	O
distributed	O
between	O
two	O
proteins	O
.	O

Iron	B-chemical
is	O
essential	O
for	O
life	O
as	O
it	O
is	O
a	O
key	O
component	O
of	O
many	O
different	O
enzymes	O
that	O
participate	O
in	O
processes	O
such	O
as	O
energy	O
production	O
and	O
metabolism	O
.	O

However	O
,	O
iron	B-chemical
can	O
also	O
be	O
highly	O
toxic	O
to	O
cells	O
because	O
it	O
readily	O
reacts	O
with	O
oxygen	B-chemical
.	O

To	O
balance	O
the	O
cell	O
’	O
s	O
need	O
for	O
iron	B-chemical
against	O
its	O
potential	O
damaging	O
effects	O
,	O
organisms	O
have	O
evolved	O
iron	B-protein_type
storage	I-protein_type
proteins	I-protein_type
known	O
as	O
ferritins	B-protein_type
that	O
form	O
cage	B-structure_element
-	I-structure_element
like	I-structure_element
structures	I-structure_element
.	O

The	O
ferritins	B-protein_type
convert	O
iron	B-chemical
into	O
a	O
less	O
reactive	O
form	O
that	O
is	O
mineralised	O
and	O
safely	O
stored	O
in	O
the	O
central	B-site
cavity	I-site
of	O
the	O
ferritin	B-protein_type
cage	O
and	O
is	O
available	O
for	O
cells	O
when	O
they	O
need	O
it	O
.	O

Recently	O
,	O
a	O
new	O
family	O
of	O
ferritins	B-protein_type
known	O
as	O
encapsulated	B-protein_state
ferritins	B-protein_type
have	O
been	O
found	O
in	O
some	O
microorganisms	B-taxonomy_domain
.	O

These	O
ferritins	B-protein_type
are	O
found	O
in	O
bacterial	B-taxonomy_domain
genomes	O
with	O
a	O
gene	O
that	O
codes	O
for	O
a	O
protein	O
cage	O
called	O
an	O
encapsulin	B-protein
.	O

Although	O
the	O
structure	B-evidence
of	O
the	O
encapsulin	B-protein
cage	O
is	O
known	O
to	O
look	O
like	O
the	O
shell	B-structure_element
of	O
a	O
virus	B-taxonomy_domain
,	O
the	O
structure	B-evidence
that	O
the	O
encapsulated	B-protein_state
ferritin	B-protein_type
itself	O
forms	O
is	O
not	O
known	O
.	O

It	O
is	O
also	O
not	O
clear	O
how	O
encapsulin	B-protein
and	O
the	O
encapsulated	B-protein_state
ferritin	B-protein_type
work	O
together	O
to	O
store	O
iron	B-chemical
.	O

He	O
et	O
al	O
.	O
have	O
now	O
used	O
the	O
techniques	O
of	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
and	O
mass	B-experimental_method
spectrometry	I-experimental_method
to	O
determine	O
the	O
structure	B-evidence
of	O
the	O
encapsulated	B-protein_state
ferritin	B-protein_type
found	O
in	O
some	O
bacteria	B-taxonomy_domain
.	O

The	O
encapsulated	B-protein_state
ferritin	B-protein_type
forms	O
a	O
ring	B-structure_element
-	I-structure_element
shaped	I-structure_element
doughnut	B-structure_element
in	O
which	O
ten	O
subunits	B-structure_element
of	O
ferritin	B-protein_type
are	O
arranged	O
in	O
a	O
ring	B-structure_element
;	O
this	O
is	O
totally	O
different	O
from	O
the	O
enclosed	O
cages	B-structure_element
that	O
other	O
ferritins	B-protein_type
form	O
.	O

Biochemical	B-experimental_method
studies	I-experimental_method
revealed	O
that	O
the	O
encapsulated	B-protein_state
ferritin	B-protein_type
is	O
able	O
to	O
convert	O
iron	B-chemical
into	O
a	O
less	O
reactive	O
form	O
,	O
but	O
it	O
cannot	O
store	O
iron	B-chemical
on	O
its	O
own	O
since	O
it	O
does	O
not	O
form	O
a	O
cage	O
.	O

Thus	O
,	O
the	O
encapsulated	B-protein_state
ferritin	B-protein_type
needs	O
to	O
be	O
housed	O
within	O
the	O
encapsulin	B-protein
cage	O
to	O
store	O
iron	B-chemical
.	O

Further	O
work	O
is	O
needed	O
to	O
investigate	O
how	O
iron	B-chemical
moves	O
into	O
the	O
encapsulin	B-protein
cage	O
to	O
reach	O
the	O
ferritin	B-protein_type
proteins	O
.	O

Some	O
organisms	O
have	O
both	O
standard	O
ferritin	B-protein_type
cages	O
and	O
encapsulated	B-protein_state
ferritins	B-protein_type
;	O
why	O
this	O
is	O
the	O
case	O
also	O
remains	O
to	O
be	O
discovered	O
.	O

Encapsulin	B-protein_type
nanocompartments	B-complex_assembly
are	O
a	O
family	O
of	O
proteinaceous	O
metabolic	O
compartments	O
that	O
are	O
widely	O
distributed	O
in	O
bacteria	B-taxonomy_domain
and	O
archaea	B-taxonomy_domain
.	O

They	O
share	O
a	O
common	O
architecture	O
,	O
comprising	O
an	O
icosahedral	B-protein_state
shell	B-structure_element
formed	O
by	O
the	O
oligomeric	O
assembly	O
of	O
a	O
protein	O
,	O
encapsulin	B-protein_type
,	O
that	O
is	O
structurally	O
related	O
to	O
the	O
HK97	B-taxonomy_domain
bacteriophage	I-taxonomy_domain
capsid	O
protein	O
gp5	B-protein
.	O

Gp5	B-protein
is	O
known	O
to	O
assemble	O
as	O
a	O
66	O
nm	O
diameter	O
icosahedral	B-protein_state
shell	B-structure_element
of	O
420	O
subunits	B-structure_element
.	O

In	O
contrast	O
,	O
both	O
the	O
Pyrococcus	B-species
furiosus	I-species
and	O
Myxococcus	B-species
xanthus	I-species
encapsulin	B-protein
shell	B-structure_element
-	O
proteins	O
form	O
32	O
nm	O
icosahedra	B-structure_element
with	O
180	O
subunits	B-structure_element
;	O
while	O
the	O
Thermotoga	B-species
maritima	I-species
encapsulin	B-protein
is	O
smaller	O
still	O
with	O
a	O
25	O
nm	O
,	O
60	O
-	O
subunit	O
icosahedron	B-structure_element
.	O

The	O
high	O
structural	O
similarity	O
of	O
the	O
encapsulin	B-protein_type
shell	B-structure_element
-	O
proteins	O
to	O
gp5	B-protein
suggests	O
a	O
common	O
evolutionary	O
origin	O
for	O
these	O
proteins	O
.	O

The	O
genes	O
encoding	O
encapsulin	B-protein_type
proteins	O
are	O
found	O
downstream	O
of	O
genes	O
for	O
dye	B-protein_type
-	I-protein_type
dependent	I-protein_type
peroxidase	I-protein_type
(	O
DyP	B-protein_type
)	O
family	O
enzymes	O
,	O
or	O
encapsulin	B-protein_type
-	I-protein_type
associated	I-protein_type
ferritins	I-protein_type
(	O
EncFtn	B-protein_type
).	O

Enzymes	O
in	O
the	O
DyP	B-protein_type
family	I-protein_type
are	O
active	O
against	O
polyphenolic	O
compounds	O
such	O
as	O
azo	O
dyes	O
and	O
lignin	O
breakdown	O
products	O
;	O
although	O
their	O
physiological	O
function	O
and	O
natural	O
substrates	O
are	O
not	O
known	O
.	O

Ferritin	B-protein_type
family	O
proteins	O
are	O
found	O
in	O
all	O
kingdoms	B-taxonomy_domain
and	O
have	O
a	O
wide	O
range	O
of	O
activities	O
,	O
including	O
ribonucleotide	B-protein_type
reductase	I-protein_type
,	O
protecting	O
DNA	O
from	O
oxidative	O
damage	O
,	O
and	O
iron	B-chemical
storage	O
.	O

The	O
classical	B-protein_state
iron	B-complex_assembly
storage	I-complex_assembly
ferritin	I-complex_assembly
nanocages	I-complex_assembly
are	O
found	O
in	O
all	O
kingdoms	B-taxonomy_domain
and	O
are	O
essential	O
in	O
eukaryotes	B-taxonomy_domain
;	O
they	O
play	O
a	O
central	O
role	O
in	O
iron	B-chemical
homeostasis	O
,	O
where	O
they	O
protect	O
the	O
cell	O
from	O
toxic	O
free	O
Fe2	B-chemical
+	I-chemical
by	O
oxidizing	O
it	O
and	O
storing	O
the	O
resulting	O
Fe3	B-chemical
+	I-chemical
as	O
ferrihydrite	B-chemical
minerals	O
within	O
their	O
central	B-site
cavity	I-site
.	O

The	O
encapsulin	B-protein_type
-	O
associated	O
enzymes	O
are	O
sequestered	O
within	O
the	O
icosahedral	B-protein_state
shell	B-structure_element
through	O
interactions	O
between	O
the	O
shell	B-structure_element
’	O
s	O
inner	O
surface	O
and	O
a	O
short	B-structure_element
localization	I-structure_element
sequence	I-structure_element
(	O
Gly	B-structure_element
-	I-structure_element
Ser	I-structure_element
-	I-structure_element
Leu	I-structure_element
-	I-structure_element
Lys	I-structure_element
)	O
appended	O
to	O
their	O
C	O
-	O
termini	O
.	O

This	B-structure_element
motif	I-structure_element
is	O
well	B-protein_state
-	I-protein_state
conserved	I-protein_state
,	O
and	O
the	O
addition	O
of	O
this	O
sequence	O
to	O
heterologous	O
proteins	O
is	O
sufficient	O
to	O
direct	O
them	O
to	O
the	O
interior	O
of	O
encapsulins	B-protein_type
.	O

A	O
recent	O
study	O
of	O
the	O
Myxococcus	B-species
xanthus	I-species
encapsulin	B-protein
showed	O
that	O
it	O
sequesters	O
a	O
number	O
of	O
different	O
EncFtn	B-protein_type
proteins	O
and	O
acts	O
as	O
an	O
‘	O
iron	B-chemical
-	O
megastore	O
’	O
to	O
protect	O
these	O
bacteria	B-taxonomy_domain
from	O
oxidative	O
stress	O
.	O

At	O
32	O
nm	O
in	O
diameter	O
,	O
it	O
is	O
much	O
larger	O
than	O
other	O
members	O
of	O
the	O
ferritin	B-protein_type
superfamily	O
,	O
such	O
as	O
the	O
12	O
nm	O
24	O
-	O
subunit	O
classical	B-protein_state
ferritin	B-protein_type
nanocage	B-complex_assembly
and	O
the	O
8	O
nm	O
12	O
-	O
subunit	O
Dps	B-protein_type
(	O
DNA	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
from	O
starved	O
cells	O
)	O
complex	O
;	O
and	O
is	O
thus	O
capable	O
of	O
sequestering	O
up	O
to	O
ten	O
times	O
more	O
iron	B-chemical
than	O
these	O
ferritins	B-protein_type
.	O

The	O
primary	O
sequences	O
of	O
EncFtn	B-protein_type
proteins	O
have	O
Glu	B-structure_element
-	I-structure_element
X	I-structure_element
-	I-structure_element
X	I-structure_element
-	I-structure_element
His	I-structure_element
metal	B-site
coordination	I-site
sites	I-site
,	O
which	O
are	O
shared	O
features	O
of	O
the	O
ferritin	B-protein_type
family	O
proteins	O
.	O

Secondary	B-experimental_method
structure	I-experimental_method
prediction	I-experimental_method
identifies	O
two	O
major	B-structure_element
α	I-structure_element
-	I-structure_element
helical	I-structure_element
regions	I-structure_element
in	O
these	O
proteins	O
;	O
this	O
is	O
in	O
contrast	O
to	O
other	O
members	O
of	O
the	O
ferritin	B-protein_type
superfamily	O
,	O
which	O
have	O
four	O
major	B-structure_element
α	I-structure_element
-	I-structure_element
helices	I-structure_element
(	O
Supplementary	O
file	O
1	O
).	O

The	O
‘	O
half	O
-	O
ferritin	B-protein_type
’	O
primary	O
sequence	O
of	O
the	O
EncFtn	B-protein_type
family	O
and	O
their	O
association	O
with	O
encapsulin	B-protein
nanocompartments	B-complex_assembly
suggests	O
a	O
distinct	O
biochemical	O
and	O
structural	O
organization	O
to	O
other	O
ferritin	B-protein_type
family	O
proteins	O
.	O

The	O
Rhodospirillum	B-species
rubrum	I-species
EncFtn	B-protein
protein	O
(	O
Rru_A0973	B-gene
)	O
shares	O
33	O
%	O
protein	O
sequence	O
identity	O
with	O
the	O
M	B-species
.	I-species
xanthus	I-species
(	O
MXAN_4464	B-gene
),	O
53	O
%	O
with	O
the	O
T	B-species
.	I-species
maritima	I-species
(	O
Tmari_0787	B-gene
),	O
and	O
29	O
%	O
with	O
the	O
P	B-species
.	I-species
furiosus	I-species
(	O
PF1192	B-gene
)	O
homologues	O
.	O

The	O
GXXH	B-structure_element
motifs	O
are	O
strictly	B-protein_state
conserved	I-protein_state
in	O
each	O
of	O
these	O
species	O
(	O
Supplementary	O
file	O
1	O
).	O

Here	O
we	O
investigate	O
the	O
structure	B-evidence
and	O
biochemistry	O
of	O
EncFtn	B-protein
in	O
order	O
to	O
understand	O
iron	B-chemical
storage	O
within	O
the	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
.	O

We	O
have	O
produced	O
recombinant	O
encapsulin	B-protein
(	O
Enc	B-protein
)	O
and	O
EncFtn	B-protein
from	O
the	O
aquatic	B-taxonomy_domain
purple	B-taxonomy_domain
-	I-taxonomy_domain
sulfur	I-taxonomy_domain
bacterium	I-taxonomy_domain
R	B-species
.	I-species
rubrum	I-species
,	O
which	O
serves	O
as	O
a	O
model	O
organism	O
for	O
the	O
study	O
of	O
the	O
control	O
of	O
the	O
bacterial	B-taxonomy_domain
nitrogen	O
fixation	O
machinery	O
,	O
in	O
Escherichia	B-species
coli	I-species
.	O

Analysis	O
by	O
transmission	B-experimental_method
electron	I-experimental_method
microscopy	I-experimental_method
(	O
TEM	B-experimental_method
)	O
indicates	O
that	O
their	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
leads	O
to	O
the	O
production	O
of	O
an	O
icosahedral	B-protein_state
nanocompartment	B-complex_assembly
with	O
encapsulated	B-protein_state
EncFtn	B-protein
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
a	O
truncated	B-protein_state
hexahistidine	B-protein_state
-	I-protein_state
tagged	I-protein_state
variant	O
of	O
the	O
EncFtn	B-protein
protein	O
(	O
EncFtnsH	B-protein
)	O
shows	O
that	O
it	O
forms	O
a	O
decameric	B-oligomeric_state
structure	B-evidence
with	O
an	O
annular	O
‘	O
ring	B-structure_element
-	I-structure_element
doughnut	I-structure_element
’	O
topology	O
,	O
which	O
is	O
distinct	O
from	O
the	O
four	B-structure_element
-	I-structure_element
helical	I-structure_element
bundles	I-structure_element
of	O
the	O
24meric	B-oligomeric_state
ferritins	B-protein_type
and	O
dodecahedral	B-oligomeric_state
DPS	B-protein_type
proteins	O
.	O

We	O
identify	O
a	O
symmetrical	O
iron	B-protein_state
bound	I-protein_state
ferroxidase	B-site
center	I-site
(	O
FOC	B-site
)	O
formed	O
between	O
subunits	B-structure_element
in	O
the	O
decamer	B-oligomeric_state
and	O
additional	O
metal	B-site
-	I-site
binding	I-site
sites	I-site
close	O
to	O
the	O
center	O
of	O
the	O
ring	B-structure_element
and	O
on	O
the	O
outer	O
surface	O
.	O

We	O
also	O
demonstrate	O
the	O
metal	O
-	O
dependent	O
assembly	O
of	O
EncFtn	B-protein
decamers	B-oligomeric_state
using	O
native	B-experimental_method
PAGE	I-experimental_method
,	O
analytical	B-experimental_method
gel	I-experimental_method
-	I-experimental_method
filtration	I-experimental_method
,	O
and	O
native	B-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
.	O

Biochemical	B-experimental_method
assays	I-experimental_method
show	O
that	O
EncFtn	B-protein
is	O
active	B-protein_state
as	O
a	O
ferroxidase	B-protein_type
enzyme	O
.	O

Through	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
we	O
show	O
that	O
the	O
conserved	B-protein_state
glutamic	B-residue_name
acid	I-residue_name
and	O
histidine	B-residue_name
residues	O
in	O
the	O
FOC	B-site
influence	O
protein	O
assembly	O
and	O
activity	O
.	O

We	O
use	O
our	O
combined	O
structural	B-evidence
and	I-evidence
biochemical	I-evidence
data	I-evidence
to	O
propose	O
a	O
model	O
for	O
the	O
EncFtn	B-protein
-	O
catalyzed	O
sequestration	O
of	O
iron	B-chemical
within	O
the	O
encapsulin	B-protein
shell	B-structure_element
.	O

Assembly	O
of	O
R	B-species
.	I-species
rubrum	I-species
EncFtn	B-protein
encapsulin	B-protein
nanocompartments	B-complex_assembly
in	O
E	B-species
.	I-species
coli	I-species

Full	B-evidence
-	I-evidence
frame	I-evidence
transmission	I-evidence
electron	I-evidence
micrographs	I-evidence
of	O
R	B-species
.	I-species
rubrum	I-species
nanocompartments	B-complex_assembly
.	O

(	O
A	O
/	O
B	O
)	O
Negative	B-experimental_method
stain	I-experimental_method
TEM	I-experimental_method
image	B-evidence
of	O
recombinant	O
R	B-species
.	I-species
rubrum	I-species
encapsulin	B-protein
and	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartments	B-complex_assembly
.	O

All	O
samples	O
were	O
imaged	O
at	O
143	O
,	O
000	O
x	O
magnification	O
;	O
the	O
scale	O
bar	O
length	O
corresponds	O
to	O
50	O
nm	O
.	O
(	O
C	O
)	O
Histogram	B-evidence
showing	O
the	O
distribution	O
of	O
nanocompartment	B-complex_assembly
diameters	O
.	O

A	O
model	O
Gaussian	B-experimental_method
nonlinear	I-experimental_method
least	I-experimental_method
square	I-experimental_method
function	I-experimental_method
was	O
fitted	O
to	O
the	O
data	O
to	O
obtain	O
a	O
mean	O
diameter	O
of	O
24	O
.	O
6	O
nm	O
with	O
a	O
standard	O
deviation	O
of	O
2	O
.	O
0	O
nm	O
for	O
encapsulin	B-protein
(	O
grey	O
)	O
and	O
a	O
mean	O
value	O
of	O
23	O
.	O
9	O
nm	O
with	O
a	O
standard	O
deviation	O
of	O
2	O
.	O
2	O
nm	O
for	O
co	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
EncFtn	B-protein
and	O
encapsulin	B-protein
(	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
,	O
black	O
).	O

Purification	O
of	O
recombinant	O
R	B-species
.	I-species
rubrum	I-species
encapsulin	B-protein
nanocompartments	B-complex_assembly
.	O

(	O
A	O
)	O
Recombinantly	B-experimental_method
expressed	I-experimental_method
encapsulin	B-protein
(	O
Enc	B-protein
)	O
and	O
co	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
were	O
purified	O
by	O
sucrose	B-experimental_method
gradient	I-experimental_method
ultracentrifugation	I-experimental_method
from	O
E	B-species
.	I-species
coli	I-species
B834	O
(	O
DE3	O
)	O
grown	O
in	O
SeMet	B-chemical
medium	O
.	O

Samples	O
were	O
resolved	O
by	O
18	O
%	O
acrylamide	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
;	O
the	O
position	O
of	O
the	O
proteins	O
found	O
in	O
the	O
complexes	O
as	O
resolved	O
on	O
the	O
gel	O
are	O
shown	O
with	O
arrows	O
.	O

(	O
B	O
/	O
C	O
)	O
Negative	B-experimental_method
stain	I-experimental_method
TEM	I-experimental_method
image	O
of	O
recombinant	O
encapsulin	B-protein
and	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartments	B-complex_assembly
.	O

Representative	O
encapsulin	B-protein
and	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
complexes	O
are	O
indicated	O
with	O
red	O
arrows	O
.	O

We	O
produced	O
recombinant	O
R	B-species
.	I-species
rubrum	I-species
encapsulin	B-protein
nanocompartments	B-complex_assembly
in	O
E	B-species
.	I-species
coli	I-species
by	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
the	O
encapsulin	B-protein
(	O
Rru_A0974	B-gene
)	O
and	O
EncFtn	B-protein
(	O
Rru_A0973	B-gene
)	O
proteins	O
,	O
and	O
purified	O
these	O
by	O
sucrose	B-experimental_method
gradient	I-experimental_method
ultra	I-experimental_method
-	I-experimental_method
centrifugation	I-experimental_method
(	O
Figure	O
1A	O
).	O

TEM	B-experimental_method
imaging	O
of	O
uranyl	O
acetate	O
-	O
stained	O
samples	O
revealed	O
that	O
,	O
when	O
expressed	B-experimental_method
in	I-experimental_method
isolation	I-experimental_method
,	O
the	O
encapsulin	B-protein
protein	O
forms	O
empty	B-protein_state
compartments	B-complex_assembly
with	O
an	O
average	O
diameter	O
of	O
24	O
nm	O
(	O
Figure	O
1B	O
and	O
Figure	O
1	O
—	O
figure	O
supplement	O
1A	O
/	O
C	O
),	O
consistent	O
with	O
the	O
appearance	O
and	O
size	O
of	O
the	O
T	B-species
.	I-species
maritima	I-species
encapsulin	B-protein
.	O

We	O
were	O
not	O
able	O
to	O
resolve	O
any	O
higher	O
-	O
order	O
structures	O
of	O
EncFtn	B-protein
by	O
TEM	B-experimental_method
.	O

Protein	O
purified	O
from	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
the	O
encapsulin	B-protein
and	O
EncFtn	B-protein
resulted	O
in	O
24	O
nm	O
compartments	O
with	O
regions	O
in	O
the	O
center	O
that	O
exclude	O
stain	O
,	O
consistent	O
with	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
EncFtn	B-protein
within	O
the	O
encapsulin	B-protein
shell	B-structure_element
(	O
Figure	O
1C	O
and	O
Figure	O
1	O
—	O
figure	O
supplement	O
1B	O
/	O
C	O
).	O

R	B-species
.	I-species
rubrum	I-species
EncFtn	B-protein
forms	O
a	O
metal	O
-	O
ion	O
stabilized	O
decamer	B-oligomeric_state
in	O
solution	O

Purification	B-experimental_method
of	I-experimental_method
recombinant	I-experimental_method
R	B-species
.	I-species
rubrum	I-species
EncFtnsH	B-protein
.	O

(	O
A	O
)	O
Recombinant	O
SeMet	B-protein_state
-	I-protein_state
labeled	I-protein_state
EncFtnsH	B-protein
produced	O
with	O
1	O
mM	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
in	O
the	O
growth	O
medium	O
was	O
purified	O
by	O
nickel	B-experimental_method
affinity	I-experimental_method
chromatography	I-experimental_method
and	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
using	O
a	O
Superdex	O
200	O
16	O
/	O
60	O
column	O
(	O
GE	O
Healthcare	O
).	O

Chromatogram	B-evidence
traces	O
measured	O
at	O
280	O
nm	O
and	O
315	O
nm	O
are	O
shown	O
with	O
the	O
results	O
from	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
analysis	O
of	O
the	O
iron	B-chemical
content	O
of	O
the	O
fractions	O
collected	O
during	O
the	O
experiment	O
.	O

The	O
peak	O
around	O
73	O
ml	O
corresponds	O
to	O
a	O
molecular	B-evidence
weight	I-evidence
of	O
around	O
130	O
kDa	O
when	O
compared	O
to	O
calibration	O
standards	O
;	O
this	O
is	O
consistent	O
with	O
a	O
decamer	B-oligomeric_state
of	O
EncFtnsH	B-protein
.	O
The	O
small	O
peak	O
at	O
85	O
ml	O
corresponds	O
to	O
the	O
13	O
kDa	O
monomer	B-oligomeric_state
compared	O
to	O
the	O
standards	O
.	O

Only	O
the	O
decamer	B-oligomeric_state
peak	O
contains	O
significant	O
amounts	O
of	O
iron	B-chemical
as	O
indicated	O
by	O
the	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
analysis	O
.	O

(	O
B	O
)	O
Peak	O
fractions	O
from	O
the	O
gel	B-experimental_method
filtration	I-experimental_method
run	O
were	O
resolved	O
by	O
15	O
%	O
acrylamide	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
and	O
stained	O
with	O
Coomassie	O
blue	O
stain	O
.	O

The	O
bands	O
around	O
13	O
kDa	O
and	O
26	O
kDa	O
correspond	O
to	O
EncFtnsH	B-protein
,	O
as	O
identified	O
by	O
MALDI	B-experimental_method
peptide	I-experimental_method
mass	I-experimental_method
fingerprinting	I-experimental_method
.	O

The	O
band	O
at	O
13	O
kDa	O
is	O
consistent	O
with	O
the	O
monomer	B-oligomeric_state
mass	O
,	O
while	O
the	O
band	O
at	O
26	O
kDa	O
is	O
consistent	O
with	O
a	O
dimer	B-oligomeric_state
of	O
EncFtnsH	B-protein
.	O
The	O
dimer	B-oligomeric_state
species	O
only	O
appears	O
in	O
the	O
decamer	B-oligomeric_state
fractions	O
.	O

(	O
C	O
)	O
SEC	B-experimental_method
-	I-experimental_method
MALLS	I-experimental_method
analysis	O
of	O
EncFtnsH	B-protein
from	O
decamer	B-oligomeric_state
fractions	O
and	O
monomer	B-oligomeric_state
fractions	O
allows	O
assignment	O
of	O
an	O
average	O
mass	O
of	O
132	O
kDa	O
to	O
decamer	B-oligomeric_state
fractions	O
and	O
13	O
kDa	O
to	O
monomer	B-oligomeric_state
fractions	O
,	O
consistent	O
with	O
decamer	B-oligomeric_state
and	O
monomer	B-oligomeric_state
species	O
(	O
Table	O
2	O
).	O

Determination	O
of	O
the	O
Fe	B-chemical
/	O
EncFtnsH	B-protein
protein	O
ratio	O
by	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
.	O

EncFtnsH	B-protein
was	O
purified	O
as	O
a	O
SeMet	B-chemical
derivative	O
from	O
E	B-species
.	I-species
coli	I-species
B834	I-species
(	I-species
DE3	I-species
)	I-species
cells	O
grown	O
in	O
SeMet	B-chemical
medium	O
with	O
1	O
mM	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
.	O

Fractions	O
from	O
SEC	B-experimental_method
were	O
collected	O
,	O
acidified	O
and	O
analysed	O
by	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
.	O

EncFtnsH	B-protein
concentration	O
was	O
calculated	O
based	O
on	O
the	O
presence	B-protein_state
of	I-protein_state
two	O
SeMet	B-chemical
per	O
mature	B-protein_state
monomer	B-oligomeric_state
.	O

These	O
data	O
were	O
collected	O
from	O
EncFtnsH	B-protein
fractions	O
from	O
a	O
single	O
gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
run	O
.	O

Peak	O
EncFtnsHretention	B-protein
volume	O
(	O
ml	O
)	O
Element	O
concentration	O
(	O
µM	O
)	O
Derived	O
EncFtnsHconcentration	B-protein
(	O
µM	O
)	O
Derived	O
Fe	B-chemical
/	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
Ca	B-chemical
Fe	B-chemical
Zn	B-chemical
Se	B-chemical
Decamer	B-oligomeric_state
66	O
.	O
5	O
n	O
.	O
d	O
.	O

Estimates	O
of	O
EncFtnsH	B-protein
molecular	B-evidence
weight	I-evidence
from	O
SEC	B-experimental_method
-	I-experimental_method
MALLS	I-experimental_method
analysis	O
.	O

EncFtnsH	B-protein
was	O
purified	O
from	O
E	B-species
.	I-species
coli	I-species
BL21	I-species
(	I-species
DE3	I-species
)	I-species
grown	O
in	O
minimal	B-experimental_method
medium	I-experimental_method
(	O
MM	B-experimental_method
)	O
by	O
nickel	B-experimental_method
affinity	I-experimental_method
chromatography	I-experimental_method
and	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
.	O

Fractions	O
from	O
two	O
peaks	B-evidence
(	O
decamer	B-oligomeric_state
and	O
monomer	B-oligomeric_state
)	O
were	O
pooled	O
separately	O
(	O
Figure	O
1C	O
)	O
and	O
analysed	O
by	O
SEC	B-experimental_method
-	I-experimental_method
MALLS	I-experimental_method
using	O
a	O
Superdex	O
200	O
10	O
/	O
300	O
GL	O
column	O
(	O
GE	O
Healthcare	O
)	O
and	O
Viscotek	O
SEC	B-experimental_method
-	I-experimental_method
MALLS	I-experimental_method
instruments	O
(	O
Malvern	O
Instruments	O
)	O
(	O
Figure	O
2C	O
).	O

The	O
decamer	B-oligomeric_state
and	O
monomer	B-oligomeric_state
peaks	B-evidence
were	O
both	O
symmetric	O
and	O
monodisperse	O
,	O
allowing	O
the	O
estimation	O
of	O
the	O
molecular	B-evidence
weight	I-evidence
of	O
the	O
species	O
in	O
these	O
fractions	O
.	O

The	O
proteins	O
analyzed	O
by	O
SEC	B-experimental_method
-	I-experimental_method
MALLS	I-experimental_method
came	O
from	O
single	O
protein	O
preparation	O
.	O

Molecular	B-evidence
Weight	I-evidence
(	O
kDa	O
)	O
Decamer	B-oligomeric_state
peak	O
Monomer	B-oligomeric_state
peak	O
Theoretical	O
133	O
13	O
EncFtnsH	B-protein
-	O
decamer	B-oligomeric_state
fractions	O
132	O
15	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
fractions	O
126	O
13	O

We	O
purified	O
recombinant	O
R	B-species
.	I-species
rubrum	I-species
EncFtn	B-protein
as	O
both	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
sequence	O
(	O
140	B-residue_range
amino	I-residue_range
acids	I-residue_range
)	O
and	O
a	O
truncated	B-protein_state
C	O
-	O
terminal	O
hexahistidine	B-protein_state
-	I-protein_state
tagged	I-protein_state
variant	O
(	O
amino	O
acids	O
1	B-residue_range
–	I-residue_range
96	I-residue_range
plus	O
the	O
tag	O
;	O
herein	O
EncFtnsH	B-protein
).	O

In	O
both	O
cases	O
the	O
elution	B-evidence
profile	I-evidence
from	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
(	O
SEC	B-experimental_method
)	O
displayed	O
two	O
peaks	B-evidence
(	O
Figure	O
2A	O
).	O

SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
analysis	O
of	O
fractions	O
from	O
these	O
peaks	B-evidence
showed	O
that	O
the	O
high	O
molecular	B-evidence
weight	I-evidence
peak	O
was	O
partially	O
resistant	O
to	O
SDS	O
and	O
heat	O
-	O
induced	O
denaturation	O
;	O
in	O
contrast	O
,	O
the	O
low	O
molecular	B-evidence
weight	I-evidence
peak	O
was	O
consistent	O
with	O
monomeric	B-oligomeric_state
mass	O
of	O
13	O
kDa	O
(	O
Figure	O
2B	O
).	O

MALDI	B-experimental_method
peptide	I-experimental_method
mass	I-experimental_method
fingerprinting	I-experimental_method
of	O
these	O
bands	O
confirmed	O
the	O
identity	O
of	O
both	O
as	O
EncFtn	B-protein
.	O

Inductively	B-experimental_method
coupled	I-experimental_method
plasma	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
(	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
)	O
analysis	O
of	O
the	O
SEC	B-experimental_method
fractions	O
showed	O
100	O
times	O
more	O
iron	B-chemical
in	O
the	O
oligomeric	O
fraction	O
than	O
the	O
monomer	B-oligomeric_state
(	O
Figure	O
2A	O
,	O
blue	O
scatter	O
points	O
;	O
Table	O
1	O
),	O
suggesting	O
that	O
EncFtn	B-protein
oligomerization	O
is	O
associated	O
with	O
iron	B-chemical
binding	O
.	O

In	O
order	O
to	O
determine	O
the	O
iron	B-chemical
-	O
loading	O
stoichiometry	O
in	O
the	O
EncFtn	B-protein
complex	O
,	O
further	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
experiments	O
were	O
performed	O
using	O
selenomethionine	B-chemical
(	O
SeMet	B-chemical
)-	O
labelled	O
protein	O
EncFtn	B-protein
(	O
Table	O
1	O
).	O

In	O
these	O
experiments	O
,	O
we	O
observed	O
sub	O
-	O
stoichiometric	O
metal	O
binding	O
,	O
which	O
is	O
in	O
contrast	O
to	O
the	O
classical	B-protein_state
ferritins	B-protein_type
.	O

Size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
with	O
multi	B-experimental_method
-	I-experimental_method
angle	I-experimental_method
laser	I-experimental_method
light	I-experimental_method
scattering	I-experimental_method
(	O
SEC	B-experimental_method
-	I-experimental_method
MALLS	I-experimental_method
)	O
analysis	O
of	O
samples	O
taken	O
from	O
each	O
peak	O
gave	O
calculated	O
molecular	O
weights	O
consistent	O
with	O
a	O
decamer	B-oligomeric_state
for	O
the	O
high	O
molecular	B-evidence
weight	I-evidence
peak	O
and	O
a	O
monomer	B-oligomeric_state
for	O
the	O
low	O
molecular	B-evidence
weight	I-evidence
peak	O
(	O
Figure	O
2C	O
,	O
Table	O
2	O
).	O

Effect	O
of	O
metal	O
ions	O
on	O
the	O
oligomeric	O
state	O
of	O
EncFtnsH	B-protein
in	O
solution	O
.	O

(	O
A	O
/	O
B	O
)	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
was	O
incubated	B-experimental_method
with	O
one	O
mole	O
equivalent	O
of	O
various	O
metal	O
salts	O
for	O
two	O
hours	O
prior	O
to	O
analytical	B-experimental_method
gel	I-experimental_method
-	I-experimental_method
filtration	I-experimental_method
using	O
a	O
Superdex	O
200	O
PC	O
3	O
.	O
2	O
/	O
30	O
column	O
.	O

Co2	B-chemical
+	I-chemical
and	O
Zn2	B-chemical
+	I-chemical
induced	O
the	O
formation	O
of	O
the	O
decameric	B-oligomeric_state
form	O
of	O
EncFtnsH	B-protein
;	O
while	O
Mn2	B-chemical
+,	I-chemical
Mg2	B-chemical
+	I-chemical
and	O
Fe3	B-chemical
+	I-chemical
did	O
not	O
significantly	O
alter	O
the	O
oligomeric	O
state	O
of	O
EncFtnsH	B-protein
.	O

PAGE	B-experimental_method
analysis	O
of	O
the	O
effect	O
of	O
metal	O
ions	O
on	O
the	O
oligomeric	O
state	O
of	O
EncFtnsH	B-protein
.	O

50	O
µM	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
or	O
decamer	B-oligomeric_state
samples	O
were	O
mixed	O
with	O
equal	O
molar	O
metal	O
ions	O
including	O
Fe2	B-chemical
+,	I-chemical
Co2	B-chemical
+,	I-chemical
Zn2	B-chemical
+,	I-chemical
Mn2	B-chemical
+,	I-chemical
Ca2	B-chemical
+,	I-chemical
Mg2	B-chemical
+	I-chemical
and	O
Fe3	B-chemical
+,	I-chemical
which	O
were	O
analyzed	O
by	O
Native	B-experimental_method
PAGE	I-experimental_method
alongside	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
.	O

(	O
A	O
)	O
10	O
%	O
Native	B-experimental_method
PAGE	I-experimental_method
analysis	O
of	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
fractions	O
mixed	O
with	O
various	O
metal	O
solutions	O
;	O
(	O
B	O
)	O
10	O
%	O
Native	B-experimental_method
PAGE	I-experimental_method
analysis	O
of	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
fractions	O
mixed	O
with	O
various	O
metal	O
solutions	O
;	O
(	O
C	O
)	O
15	O
%	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
analysis	O
on	O
the	O
mixtures	O
of	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
fractions	O
and	O
metal	O
solutions	O
;	O
(	O
D	O
)	O
15	O
%	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
analysis	O
on	O
the	O
mixtures	O
of	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
fractions	O
and	O
metal	O
solutions	O
.	O

Effect	O
of	O
Fe2	B-chemical
+	I-chemical
and	O
protein	O
concentration	O
on	O
the	O
oligomeric	O
state	O
of	O
EncFtnsH	B-protein
in	O
solution	O
.	O

(	O
A	O
)	O
Recombinant	O
EncFtnsH	B-protein
was	O
purified	O
by	O
Gel	B-experimental_method
filtration	I-experimental_method
Superdex	O
200	O
chromatography	O
from	O
E	B-species
.	I-species
coli	I-species
BL21	I-species
(	I-species
DE3	I-species
)	I-species
grown	O
in	O
MM	B-experimental_method
or	O
in	O
MM	B-experimental_method
supplemented	O
with	O
1	O
mM	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
(	O
MM	B-experimental_method
+	O
Fe2	B-chemical
+).	I-chemical

A	O
higher	O
proportion	O
of	O
decamer	B-oligomeric_state
(	O
peak	O
between	O
65	O
and	O
75	O
ml	O
)	O
is	O
seen	O
in	O
the	O
sample	O
purified	O
from	O
MM	B-experimental_method
+	O
Fe2	B-chemical
+	I-chemical
compared	O
to	O
EncFtnsH	B-protein
-	O
MM	B-experimental_method
,	O
indicating	O
that	O
Fe2	B-chemical
+	I-chemical
facilitates	O
the	O
multimerization	O
of	O
EncFtnsH	B-protein
in	O
vivo	O
.	O
(	O
B	O
)	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
was	O
incubated	O
with	O
one	O
molar	O
equivalent	O
of	O
Fe2	B-chemical
+	I-chemical
salts	O
for	O
two	O
hours	O
prior	O
to	O
analytical	B-experimental_method
gel	I-experimental_method
-	I-experimental_method
filtration	I-experimental_method
using	O
a	O
Superdex	O
200	O
PC	O
3	O
.	O
2	O
/	O
30	O
column	O
(	O
GE	O
Healthcare	O
).	O

Both	O
Fe2	B-chemical
+	I-chemical
salts	O
tested	O
induced	O
the	O
formation	O
of	O
decamer	B-oligomeric_state
indicated	O
by	O
the	O
peak	O
between	O
1	O
.	O
2	O
and	O
1	O
.	O
6	O
ml	O
.	O

Monomeric	B-oligomeric_state
and	O
decameric	B-oligomeric_state
samples	O
of	O
EncFtnsH	B-protein
are	O
shown	O
as	O
controls	O
.	O

Peaks	B-evidence
around	O
0	O
.	O
8	O
ml	O
were	O
seen	O
as	O
protein	O
aggregation	O
.	O

(	O
C	O
)	O
Analytical	B-experimental_method
gel	I-experimental_method
filtration	I-experimental_method
of	O
EncFtn	B-protein
monomer	B-oligomeric_state
at	O
different	O
concentrations	O
to	O
illustrate	O
the	O
effect	O
of	O
protein	O
concentration	O
on	O
multimerization	O
.	O

The	O
major	O
peak	O
shows	O
a	O
shift	O
towards	O
a	O
dimer	B-oligomeric_state
species	O
at	O
high	O
concentration	O
of	O
protein	O
,	O
but	O
the	O
ratio	O
of	O
this	O
peak	O
(	O
1	O
.	O
5	O
–	O
1	O
.	O
8	O
ml	O
)	O
to	O
the	O
decamer	B-oligomeric_state
peak	O
(	O
1	O
.	O
2	O
–	O
1	O
.	O
5	O
ml	O
)	O
does	O
not	O
change	O
when	O
compared	O
to	O
the	O
low	O
concentration	O
sample	O
.	O

Gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
peak	B-evidence
area	I-evidence
ratios	I-evidence
for	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
and	O
monomer	B-oligomeric_state
on	O
addition	O
of	O
different	O
metal	O
ions	O
.	O

EncFtnsH	B-protein
was	O
produced	O
in	O
E	B-species
.	I-species
coli	I-species
BL21	I-species
(	I-species
DE3	I-species
)	I-species
cultured	O
in	O
MM	B-experimental_method
and	O
MM	B-experimental_method
with	O
1	O
mM	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
(	O
MM	B-experimental_method
+	O
Fe2	B-chemical
+)	I-chemical
and	O
purified	O
by	O
gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
chromatography	I-experimental_method
using	O
an	O
Superdex	O
200	O
16	O
/	O
60	O
column	O
(	O
GE	O
Healthcare	O
).	O

Monomer	B-oligomeric_state
fractions	O
of	O
EncFtnsH	B-protein
purified	O
from	O
MM	B-experimental_method
were	O
pooled	O
and	O
run	O
in	O
subsequent	O
analytical	B-experimental_method
gel	I-experimental_method
-	I-experimental_method
filtration	I-experimental_method
runs	O
over	O
the	O
course	O
of	O
three	O
days	O
.	O

Samples	O
of	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
were	O
incubated	O
with	O
one	O
molar	O
equivalent	O
of	O
metal	O
ion	O
salts	O
at	O
room	O
temperature	O
for	O
two	O
hours	O
before	O
analysis	O
by	O
analytical	B-experimental_method
gel	I-experimental_method
filtration	I-experimental_method
chromatography	I-experimental_method
(	O
AGF	B-experimental_method
)	O
using	O
a	O
Superdex	O
200	O
10	O
/	O
300	O
GL	O
column	O
.	O

The	O
area	O
for	O
resulting	O
protein	O
peaks	B-evidence
were	O
calculated	O
using	O
the	O
Unicorn	O
software	O
(	O
GE	O
Healthcare	O
);	O
peak	B-evidence
ratios	I-evidence
were	O
calculated	O
to	O
quantify	O
the	O
propensity	O
of	O
EncFtnsH	B-protein
to	O
multimerize	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
different	O
metal	O
ions	O
.	O

The	O
change	O
in	O
the	O
ratios	O
of	O
monomer	B-oligomeric_state
to	O
decamer	B-oligomeric_state
over	O
the	O
three	O
days	O
of	O
experiments	O
may	O
be	O
a	O
consequence	O
of	O
experimental	O
variability	O
,	O
or	O
the	O
propensity	O
of	O
this	O
protein	O
to	O
equilibrate	O
towards	O
decamer	B-oligomeric_state
over	O
time	O
.	O

The	O
increased	O
decamer	B-oligomeric_state
:	O
monomer	B-oligomeric_state
ratio	O
seen	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Fe2	B-chemical
+,	I-chemical
Co2	B-chemical
+,	I-chemical
and	O
Zn2	B-chemical
+	I-chemical
indicates	O
that	O
these	O
metal	O
ions	O
facilitate	O
multimerization	O
of	O
the	O
EncFtnsH	B-protein
protein	O
,	O
while	O
the	O
other	O
metal	O
ions	O
tested	O
do	O
not	O
appear	O
to	O
induce	O
multimerization	O
.	O

The	O
analytical	B-experimental_method
gel	I-experimental_method
filtration	I-experimental_method
experiment	O
was	O
repeated	O
twice	O
using	O
two	O
independent	O
preparations	O
of	O
protein	O
,	O
of	O
which	O
values	O
calculated	O
from	O
one	O
sample	O
are	O
presented	O
here	O
.	O

Method	O
Sample	O
Monomer	B-oligomeric_state
area	O
Decamer	B-oligomeric_state
area	O
Decamer	B-oligomeric_state
/	O
Monomer	B-oligomeric_state
Gel	B-experimental_method
filtration	I-experimental_method
Superdex	O
200	O
chromatography	O
EncFtnsH	B-protein
-	O
MM	B-experimental_method
64	O
.	O
3	O
583	O
.	O
6	O
0	O
.	O
1	O
EncFtnsH	B-protein
-	O
MM	B-experimental_method
+	O
Fe2	B-chemical
+	I-chemical
1938	O
.	O
4	O
426	O
.	O
4	O
4	O
.	O
5	O
Analytical	B-experimental_method
Gel	I-experimental_method
filtration	I-experimental_method
Day1	O
EncFtnsH	B-protein
-	O
decamer	B-oligomeric_state
fractions	O
20	O
.	O
2	O
1	O
.	O
8	O
11	O
.	O
2	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
fractions	O
2	O
.	O
9	O
21	O
.	O
9	O
0	O
.	O
1	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
11	O
.	O
0	O
13	O
.	O
0	O
0	O
.	O
8	O
FeSO4	B-chemical
-	I-chemical
HCl	I-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
11	O
.	O
3	O
11	O
.	O
4	O
1	O
.	O
0	O
Analytical	B-experimental_method
Gel	I-experimental_method
filtration	I-experimental_method
Day2	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
fractions	O
8	O
.	O
3	O
22	O
.	O
8	O
0	O
.	O
4	O
CoCl2	B-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
17	O
.	O
7	O
14	O
.	O
5	O
1	O
.	O
2	O
MnCl2	B-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
3	O
.	O
1	O
30	O
.	O
5	O
0	O
.	O
1	O
ZnSO4	B-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
20	O
.	O
4	O
9	O
.	O
0	O
2	O
.	O
3	O
FeCl3	B-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
3	O
.	O
9	O
28	O
.	O
6	O
0	O
.	O
1	O
Analytical	B-experimental_method
Gel	I-experimental_method
filtration	I-experimental_method
Day3	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
fractions	O
6	O
.	O
3	O
23	O
.	O
4	O
0	O
.	O
3	O
MgSO4	B-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
5	O
.	O
8	O
30	O
.	O
2	O
0	O
.	O
2	O
Ca	B-chemical
acetate	I-chemical
/	O
EncFtnsH	B-protein
-	O
monomer	B-oligomeric_state
5	O
.	O
6	O
25	O
.	O
2	O
0	O
.	O
2	O

We	O
purified	O
EncFtnsH	B-protein
from	O
E	B-species
.	I-species
coli	I-species
grown	O
in	O
MM	B-experimental_method
with	O
or	O
without	O
the	O
addition	O
of	O
1	O
mM	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
.	O

The	O
decamer	B-oligomeric_state
to	O
monomer	B-oligomeric_state
ratio	O
in	O
the	O
sample	O
purified	O
from	O
cells	O
grown	O
in	O
iron	B-chemical
-	O
supplemented	O
media	O
was	O
4	O
.	O
5	O
,	O
while	O
that	O
from	O
the	O
iron	B-protein_state
-	I-protein_state
free	I-protein_state
media	O
was	O
0	O
.	O
11	O
,	O
suggesting	O
that	O
iron	B-chemical
induces	O
the	O
oligomerization	O
of	O
EncFtnsH	B-protein
in	O
vivo	O
(	O
Figure	O
3A	O
,	O
Table	O
3	O
).	O

To	O
test	O
the	O
metal	O
-	O
dependent	O
oligomerization	O
of	O
EncFtnsH	B-protein
in	O
vitro	O
,	O
we	O
incubated	B-experimental_method
the	O
protein	O
with	O
various	O
metal	O
cations	O
and	O
subjected	O
samples	O
to	O
analytical	B-experimental_method
SEC	I-experimental_method
and	O
non	B-experimental_method
-	I-experimental_method
denaturing	I-experimental_method
PAGE	I-experimental_method
.	O

Of	O
the	O
metals	O
tested	O
,	O
only	O
Fe2	B-chemical
+,	I-chemical
Zn2	B-chemical
+	I-chemical
and	O
Co2	B-chemical
+	I-chemical
induced	O
the	O
formation	O
of	O
significant	O
amounts	O
of	O
the	O
decamer	B-oligomeric_state
(	O
Figure	O
3B	O
,	O
Figure	O
3	O
—	O
figure	O
supplement	O
1	O
/	O
2	O
).	O

While	O
Fe2	B-chemical
+	I-chemical
induces	O
the	O
multimerization	O
of	O
EncFtnsH	B-protein
,	O
Fe3	B-chemical
+	I-chemical
in	O
the	O
form	O
of	O
FeCl3	B-chemical
does	O
not	O
have	O
this	O
effect	O
on	O
the	O
protein	O
,	O
highlighting	O
the	O
apparent	O
preference	O
this	O
protein	O
has	O
for	O
the	O
ferrous	B-chemical
form	I-chemical
of	I-chemical
iron	I-chemical
.	O

To	O
determine	O
if	O
the	O
oligomerization	O
of	O
EncFtnsH	B-protein
was	O
concentration	O
dependent	O
we	O
performed	O
analytical	B-experimental_method
SEC	I-experimental_method
at	O
90	O
and	O
700	O
µM	O
protein	O
concentration	O
(	O
Figure	O
3C	O
).	O

At	O
the	O
higher	O
concentration	O
,	O
no	O
increase	O
in	O
the	O
decameric	B-oligomeric_state
form	O
of	O
EncFtn	B-protein
was	O
observed	O
;	O
however	O
,	O
the	O
shift	O
in	O
the	O
major	O
peak	O
from	O
the	O
position	O
of	O
the	O
monomer	B-oligomeric_state
species	O
indicated	O
a	O
tendency	O
to	O
dimerize	B-oligomeric_state
at	O
high	O
concentration	O
.	O

Crystal	B-evidence
structure	I-evidence
of	O
EncFtnsH	B-protein

Electrostatic	O
surface	O
of	O
EncFtnsH	B-protein
.	O

The	O
solvent	O
accessible	O
surface	O
of	O
EncFtnsH	B-protein
is	O
shown	O
,	O
colored	O
by	O
electrostatic	O
potential	O
as	O
calculated	O
using	O
the	O
APBS	O
plugin	O
in	O
PyMOL	O
.	O

Negatively	O
charged	O
regions	O
are	O
colored	O
red	O
and	O
positive	O
regions	O
in	O
blue	O
,	O
neutral	O
regions	O
in	O
grey	O
.	O
(	O
A	O
)	O
View	O
of	O
the	O
surface	O
of	O
the	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
looking	O
down	O
the	O
central	O
axis	O
.	O

(	O
B	O
)	O
Orthogonal	O
view	O
of	O
(	O
A	O
).	O
(	O
C	O
)	O
Cutaway	O
view	O
of	O
(	O
B	O
)	O
showing	O
the	O
charge	O
distribution	O
within	O
the	O
central	B-site
cavity	I-site
.	O

Crystal	B-evidence
structure	I-evidence
of	O
EncFtnsH	B-protein
.	O

(	O
A	O
)	O
Overall	O
architecture	O
of	O
EncFtnsH	B-protein
.	O
Transparent	O
solvent	O
accessible	O
surface	O
view	O
with	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
shown	O
as	O
tubes	O
and	O
bound	O
metal	O
ions	O
as	O
spheres	O
.	O

Alternating	O
subunits	B-structure_element
are	O
colored	O
blue	O
and	O
green	O
for	O
clarity	O
.	O

The	O
doughnut	B-structure_element
-	I-structure_element
like	I-structure_element
decamer	B-oligomeric_state
is	O
7	O
nm	O
in	O
diameter	O
and	O
4	O
.	O
5	O
nm	O
thick	O
.	O
(	O
B	O
)	O
Monomer	B-oligomeric_state
of	O
EncFtnsH	B-protein
shown	O
as	O
a	O
secondary	O
structure	O
cartoon	O
.	O
(	O
C	O
/	O
D	O
)	O
Dimer	B-site
interfaces	I-site
formed	O
in	O
the	O
decameric	B-oligomeric_state
ring	B-structure_element
of	O
EncFtnsH	B-protein
.	O
Subunits	B-structure_element
are	O
shown	O
as	O
secondary	O
structure	O
cartoons	O
and	O
colored	O
blue	O
and	O
green	O
for	O
clarity	O
.	O

Bound	O
metal	O
ions	O
are	O
shown	O
as	O
orange	O
spheres	O
for	O
Fe3	B-chemical
+	I-chemical
and	O
grey	O
and	O
white	O
spheres	O
for	O
Ca2	B-chemical
+.	I-chemical

We	O
determined	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
EncFtnsH	B-protein
by	O
molecular	B-experimental_method
replacement	I-experimental_method
to	O
2	O
.	O
0	O
Å	O
resolution	O
(	O
see	O
Table	O
1	O
for	O
X	B-evidence
-	I-evidence
ray	I-evidence
data	I-evidence
collection	I-evidence
and	I-evidence
refinement	I-evidence
statistics	I-evidence
).	O

The	O
crystallographic	O
asymmetric	O
unit	O
contained	O
thirty	O
monomers	B-oligomeric_state
of	O
EncFtn	B-protein
with	O
visible	O
electron	B-evidence
density	I-evidence
for	O
residues	O
7	B-residue_range
–	I-residue_range
96	I-residue_range
in	O
each	O
chain	O
.	O

The	O
protein	O
chains	O
were	O
arranged	O
as	O
three	O
identical	O
annular	B-structure_element
decamers	B-oligomeric_state
,	O
each	O
with	O
D5	O
symmetry	O
.	O

The	O
decamer	B-oligomeric_state
has	O
a	O
diameter	O
of	O
7	O
nm	O
and	O
thickness	O
of	O
4	O
nm	O
(	O
Figure	O
4A	O
).	O

The	O
monomer	B-oligomeric_state
of	O
EncFtn	B-protein
has	O
an	O
N	O
-	O
terminal	O
310	B-structure_element
-	I-structure_element
helix	I-structure_element
that	O
precedes	O
two	O
4	O
nm	O
long	O
antiparallel	B-structure_element
α	I-structure_element
-	I-structure_element
helices	I-structure_element
arranged	O
with	O
their	O
long	O
axes	O
at	O
25	O
°	O
to	O
each	O
other	O
;	O
these	O
helices	B-structure_element
are	O
followed	O
by	O
a	O
shorter	O
1	O
.	O
4	O
nm	O
helix	B-structure_element
projecting	O
at	O
70	O
°	O
from	O
α2	B-structure_element
(	O
Figure	O
4B	O
).	O

The	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
the	O
crystallized	O
construct	O
extends	O
from	O
the	O
outer	O
circumference	O
of	O
the	O
ring	B-structure_element
,	O
indicating	O
that	O
the	O
encapsulin	B-site
localization	I-site
sequence	I-site
in	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
is	O
on	O
the	O
exterior	O
of	O
the	O
ring	B-structure_element
and	O
is	O
thus	O
free	O
to	O
interact	O
with	O
its	O
binding	B-site
site	I-site
on	O
the	O
encapsulin	B-protein
shell	B-structure_element
protein	O
.	O

The	O
monomer	B-oligomeric_state
of	O
EncFtnsH	B-protein
forms	O
two	O
distinct	O
dimer	B-site
interfaces	I-site
within	O
the	O
decamer	B-oligomeric_state
(	O
Figure	O
4	O
C	O
/	O
D	O
).	O

The	O
first	O
dimer	B-oligomeric_state
is	O
formed	O
from	O
two	O
monomers	B-oligomeric_state
arranged	O
antiparallel	O
to	O
each	O
other	O
,	O
with	O
α1	B-structure_element
from	O
each	O
monomer	B-oligomeric_state
interacting	O
along	O
their	O
lengths	O
and	O
α3	B-structure_element
interdigitating	O
with	O
α2	B-structure_element
and	O
α3	B-structure_element
of	O
the	O
partner	O
chain	O
.	O

This	O
interface	B-site
buries	O
one	O
third	O
of	O
the	O
surface	O
area	O
from	O
each	O
partner	O
and	O
is	O
stabilized	O
by	O
thirty	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
fourteen	O
salt	B-bond_interaction
bridges	I-bond_interaction
(	O
Figure	O
4C	O
).	O

The	O
second	O
dimer	B-site
interface	I-site
forms	O
an	O
antiparallel	B-structure_element
four	I-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
between	O
helices	B-structure_element
1	I-structure_element
and	I-structure_element
2	I-structure_element
from	O
each	O
monomer	B-oligomeric_state
(	O
Figure	O
4D	O
).	O

This	O
interface	B-site
is	O
less	O
extensive	O
than	O
the	O
first	O
and	O
is	O
stabilized	O
by	O
twenty	O
-	O
one	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
,	O
six	O
salt	B-bond_interaction
bridges	I-bond_interaction
,	O
and	O
a	O
number	O
of	O
metal	O
ions	O
.	O

The	O
arrangement	O
of	O
ten	O
monomers	B-oligomeric_state
in	O
alternating	O
orientation	O
forms	O
the	O
decamer	B-oligomeric_state
of	O
EncFtn	B-protein
,	O
which	O
assembles	O
as	O
a	O
pentamer	B-oligomeric_state
of	O
dimers	B-oligomeric_state
(	O
Figure	O
4A	O
).	O

Each	O
monomer	B-oligomeric_state
lies	O
at	O
45	O
°	O
relative	O
to	O
the	O
vertical	O
central	O
-	O
axis	O
of	O
the	O
ring	B-structure_element
,	O
with	O
the	O
N	O
-	O
termini	O
of	O
alternating	O
subunits	B-structure_element
capping	O
the	O
center	O
of	O
the	O
ring	B-structure_element
at	O
each	O
end	O
,	O
while	O
the	O
C	O
-	O
termini	O
are	O
arranged	O
around	O
the	O
circumference	O
.	O

The	O
central	B-site
hole	I-site
in	O
the	O
ring	B-structure_element
is	O
2	O
.	O
5	O
nm	O
at	O
its	O
widest	O
in	O
the	O
center	O
of	O
the	O
complex	O
,	O
and	O
1	O
.	O
5	O
nm	O
at	O
its	O
narrowest	O
point	O
near	O
the	O
outer	O
surface	O
,	O
although	O
it	O
should	O
be	O
noted	O
that	O
a	O
number	O
of	O
residues	O
at	O
the	O
N	O
-	O
terminus	O
are	O
not	O
visible	O
in	O
the	O
crystallographic	B-evidence
electron	I-evidence
density	I-evidence
and	O
these	O
may	O
occupy	O
the	O
central	B-site
channel	I-site
.	O

The	O
surface	O
of	O
the	O
decamer	B-oligomeric_state
has	O
distinct	O
negatively	B-site
charged	I-site
patches	I-site
,	O
both	O
within	O
the	O
central	B-site
hole	I-site
and	O
on	O
the	O
outer	O
circumference	O
,	O
which	O
form	O
spokes	B-structure_element
through	O
the	O
radius	O
of	O
the	O
complex	O
(	O
Figure	O
4	O
—	O
figure	O
supplement	O
1	O
).	O

EncFtn	B-protein
ferroxidase	B-site
center	I-site

Putative	O
ligand	B-site
-	I-site
binding	I-site
site	I-site
in	O
EncFtnsH	B-protein
.	O

(	O
A	O
)	O
Wall	O
-	O
eyed	O
stereo	O
view	O
of	O
the	O
dimer	B-site
interface	I-site
of	O
EncFtn	B-protein
.	O

Protein	O
chains	O
are	O
shown	O
as	O
sticks	O
,	O
with	O
2mFo	B-evidence
-	I-evidence
DFc	I-evidence
electron	I-evidence
density	I-evidence
shown	O
in	O
blue	O
mesh	O
and	O
contoured	O
at	O
1	O
.	O
5	O
σ	O
and	O
mFo	B-evidence
-	I-evidence
DFc	I-evidence
shown	O
in	O
green	O
mesh	O
and	O
contoured	O
at	O
3	O
σ	O
.	O
(	O
B	O
)	O
Wall	O
-	O
eyed	O
stereo	O
view	O
of	O
putative	O
metal	B-site
binding	I-site
site	I-site
at	O
the	O
external	O
surface	O
of	O
EncFtnsH	B-protein
.	O
Protein	O
chains	O
and	O
electron	B-evidence
density	I-evidence
maps	I-evidence
are	O
shown	O
as	O
in	O
(	O
A	O
).	O

EncFtnsH	B-protein
metal	B-site
binding	I-site
sites	I-site
.	O

(	O
A	O
)	O
Wall	O
-	O
eyed	O
stereo	O
view	O
of	O
the	O
metal	B-site
-	I-site
binding	I-site
dimerization	I-site
interface	I-site
of	O
EncFtnsH	B-protein
.	O
Protein	O
residues	O
are	O
shown	O
as	O
sticks	O
with	O
blue	O
and	O
green	O
carbons	O
for	O
the	O
different	O
subunits	B-structure_element
,	O
iron	B-chemical
ions	O
are	O
shown	O
as	O
orange	O
spheres	O
and	O
calcium	B-chemical
as	O
grey	O
spheres	O
,	O
and	O
the	O
glycolic	B-chemical
acid	I-chemical
ligand	O
is	O
shown	O
with	O
yellow	O
carbon	O
atoms	O
coordinated	O
above	O
the	O
di	B-site
-	I-site
iron	I-site
center	I-site
.	O

The	O
2mFo	B-evidence
-	I-evidence
DFc	I-evidence
electron	I-evidence
density	I-evidence
map	I-evidence
is	O
shown	O
as	O
a	O
blue	O
mesh	O
contoured	O
at	O
1	O
.	O
5	O
σ	O
and	O
the	O
NCS	B-evidence
-	I-evidence
averaged	I-evidence
anomalous	I-evidence
difference	I-evidence
map	I-evidence
is	O
shown	O
as	O
an	O
orange	O
mesh	O
and	O
contoured	O
at	O
10	O
σ	O
.	O
(	O
B	O
)	O
Iron	B-chemical
coordination	B-bond_interaction
within	O
the	O
FOC	B-site
including	O
residues	O
Glu32	B-residue_name_number
,	O
Glu62	B-residue_name_number
,	O
His65	B-residue_name_number
and	O
Tyr39	B-residue_name_number
from	O
two	O
chains	O
.	O

Protein	O
and	O
metal	O
ions	O
are	O
shown	O
as	O
in	O
A	O
.	O
Coordination	B-bond_interaction
between	O
the	O
protein	O
and	O
iron	B-chemical
ions	O
is	O
shown	O
as	O
yellow	O
dashed	O
lines	O
with	O
distances	O
indicated	O
.	O
(	O
C	O
)	O
Coordination	B-bond_interaction
of	O
calcium	B-chemical
within	O
the	O
dimer	B-site
interface	I-site
by	O
four	O
glutamic	B-residue_name
acid	I-residue_name
residues	O
(	O
E31	B-residue_name_number
and	O
E34	B-residue_name_number
from	O
two	O
chains	O
).	O

The	O
calcium	B-chemical
ion	O
is	O
shown	O
as	O
a	O
grey	O
sphere	O
and	O
water	B-chemical
molecules	O
involved	O
in	O
the	O
coordination	B-bond_interaction
of	O
the	O
calcium	B-chemical
ion	O
are	O
shown	O
as	O
crosses	O
.	O
(	O
D	O
)	O
Metal	B-site
coordination	I-site
site	I-site
on	O
the	O
outer	O
surface	O
of	O
EncFtnsH	B-protein
.	O
The	O
two	O
calcium	B-chemical
ions	O
are	O
coordinated	B-bond_interaction
by	I-bond_interaction
residues	O
His57	B-residue_name_number
,	O
Glu61	B-residue_name_number
and	O
Glu64	B-residue_name_number
from	O
the	O
two	O
chains	O
of	O
the	O
FOC	B-site
dimer	B-oligomeric_state
,	O
and	O
are	O
located	O
at	O
the	O
outer	O
surface	O
of	O
the	O
complex	O
,	O
positioned	O
10	O
Å	O
away	O
from	O
the	O
FOC	B-site
iron	B-chemical
.	O

The	O
electron	B-evidence
density	I-evidence
maps	I-evidence
of	O
the	O
initial	O
EncFtnsH	B-protein
model	O
displayed	O
significant	O
positive	O
peaks	O
in	O
the	O
mFo	B-evidence
-	I-evidence
DFc	I-evidence
map	I-evidence
at	O
the	O
center	O
of	O
the	O
4	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
dimer	B-oligomeric_state
(	O
Figure	O
5	O
—	O
figure	O
supplement	O
1	O
).	O

Informed	O
by	O
the	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
data	O
indicating	O
the	O
presence	B-protein_state
of	I-protein_state
iron	B-chemical
in	O
the	O
protein	O
we	O
collected	O
diffraction	B-evidence
data	I-evidence
at	O
the	O
experimentally	O
determined	O
iron	B-chemical
absorption	O
edge	O
(	O
1	O
.	O
74	O
Å	O
)	O
and	O
calculated	O
an	O
anomalous	B-evidence
difference	I-evidence
Fourier	I-evidence
map	I-evidence
using	O
this	O
data	O
.	O

Inspection	O
of	O
this	O
map	B-evidence
showed	O
two	O
10	O
-	O
sigma	O
peaks	B-evidence
between	O
residues	O
Glu32	B-residue_name_number
,	O
Glu62	B-residue_name_number
and	O
His65	B-residue_name_number
of	O
two	O
adjacent	O
chains	O
,	O
and	O
a	O
statistically	O
smaller	O
5	O
-	O
sigma	O
peak	O
between	O
residues	O
Glu31	B-residue_name_number
and	O
Glu34	B-residue_name_number
of	O
the	O
two	O
chains	O
.	O

Modeling	O
metal	O
ions	O
into	O
these	O
peaks	O
and	O
refinement	B-experimental_method
of	O
the	O
anomalous	B-evidence
scattering	I-evidence
parameters	I-evidence
allowed	O
us	O
to	O
identify	O
these	O
as	O
two	O
iron	B-chemical
ions	O
and	O
a	O
calcium	B-chemical
ion	O
respectively	O
(	O
Figure	O
5A	O
).	O

An	O
additional	O
region	O
of	O
asymmetric	O
electron	B-evidence
density	I-evidence
near	O
the	O
di	B-site
-	I-site
iron	I-site
binding	I-site
site	I-site
in	O
the	O
mFo	B-evidence
-	I-evidence
DFc	I-evidence
map	I-evidence
was	O
modeled	O
as	O
glycolic	B-chemical
acid	I-chemical
,	O
presumably	O
a	O
breakdown	O
product	O
of	O
the	O
PEG	B-chemical
3350	I-chemical
used	O
for	O
crystallization	O
.	O

This	O
di	B-site
-	I-site
iron	I-site
center	I-site
has	O
an	O
Fe	B-evidence
-	I-evidence
Fe	I-evidence
distance	I-evidence
of	O
3	O
.	O
5	O
Å	O
,	O
Fe	B-evidence
-	I-evidence
Glu	I-evidence
-	I-evidence
O	I-evidence
distances	I-evidence
between	O
2	O
.	O
3	O
and	O
2	O
.	O
5	O
Å	O
,	O
and	O
Fe	B-evidence
-	I-evidence
His	I-evidence
-	I-evidence
N	I-evidence
distances	I-evidence
of	O
2	O
.	O
5	O
Å	O
(	O
Figure	O
5B	O
).	O

This	O
coordination	B-bond_interaction
geometry	O
is	O
consistent	O
with	O
the	O
di	B-site
-	I-site
nuclear	I-site
ferroxidase	I-site
center	I-site
(	O
FOC	B-site
)	O
found	O
in	O
ferritin	B-protein_type
.	O

It	O
is	O
interesting	O
to	O
note	O
that	O
although	O
we	O
did	O
not	O
add	O
any	O
additional	O
iron	B-chemical
to	O
the	O
crystallization	B-experimental_method
trials	I-experimental_method
,	O
the	O
FOC	B-site
was	O
fully	O
occupied	O
with	O
iron	B-chemical
in	O
the	O
final	O
structure	B-evidence
,	O
implying	O
that	O
this	O
site	O
has	O
a	O
very	O
high	O
affinity	B-evidence
for	O
iron	B-chemical
.	O

The	O
calcium	B-chemical
ion	O
coordinated	B-bond_interaction
by	I-bond_interaction
Glu31	B-residue_name_number
and	O
Glu34	B-residue_name_number
adopts	O
heptacoordinate	B-protein_state
geometry	O
,	O
with	O
coordination	B-bond_interaction
distances	O
of	O
2	O
.	O
5	O
Å	O
between	O
the	O
metal	O
ion	O
and	O
carboxylate	O
oxygens	O
of	O
Glu31	B-residue_name_number
and	O
Glu34	B-residue_name_number
(	O
E31	B-site
/	I-site
34	I-site
-	I-site
site	I-site
).	O

A	O
number	O
of	O
ordered	O
solvent	O
molecules	O
are	O
also	O
coordinated	B-bond_interaction
to	O
this	O
metal	O
ion	O
at	O
a	O
distance	O
of	O
2	O
.	O
5	O
Å	O
.	O
This	O
heptacoordinate	B-protein_state
geometry	O
is	O
common	O
in	O
crystal	B-evidence
structures	I-evidence
with	O
calcium	B-chemical
ions	O
(	O
Figure	O
5C	O
).	O

While	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
indicated	O
that	O
there	O
were	O
negligible	O
amounts	O
of	O
calcium	B-chemical
in	O
the	O
purified	O
protein	O
,	O
the	O
presence	B-protein_state
of	I-protein_state
140	O
mM	O
calcium	B-chemical
acetate	I-chemical
in	O
the	O
crystallization	O
mother	O
liquor	O
favors	O
the	O
coordination	B-bond_interaction
of	O
calcium	B-chemical
at	O
this	O
site	O
.	O

The	O
fact	O
that	O
the	O
protein	O
does	O
not	O
multimerize	O
in	O
solution	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Fe3	B-chemical
+	I-chemical
may	O
indicate	O
that	O
these	O
metal	B-site
binding	I-site
sites	I-site
have	O
a	O
lower	O
affinity	O
for	O
the	O
ferric	O
form	O
of	O
iron	B-chemical
,	O
which	O
is	O
the	O
product	O
of	O
the	O
ferroxidase	B-protein_type
reaction	O
.	O

A	O
number	O
of	O
additional	O
metal	O
-	O
ions	O
were	O
present	O
at	O
the	O
outer	O
circumference	O
of	O
at	O
least	O
one	O
decamer	B-oligomeric_state
in	O
the	O
asymmetric	O
unit	O
(	O
Figure	O
5D	O
).	O

These	O
ions	O
are	O
coordinated	B-bond_interaction
by	I-bond_interaction
His57	B-residue_name_number
,	O
Glu61	B-residue_name_number
and	O
Glu64	B-residue_name_number
from	O
both	O
chains	O
in	O
the	O
FOC	B-site
dimer	B-oligomeric_state
and	O
are	O
4	O
.	O
5	O
Å	O
apart	O
;	O
Fe	B-evidence
-	I-evidence
Glu	I-evidence
-	I-evidence
O	I-evidence
distances	O
are	O
between	O
2	O
.	O
5	O
and	O
3	O
.	O
5	O
Å	O
and	O
the	O
Fe	B-evidence
-	I-evidence
His	I-evidence
-	I-evidence
N	I-evidence
distances	I-evidence
are	O
4	O
and	O
4	O
.	O
5	O
Å	O
.	O

Comparison	O
of	O
quaternary	O
structure	O
of	O
EncFtnsH	B-protein
and	O
ferritin	B-protein_type
.	O

(	O
A	O
)	O
Aligned	B-experimental_method
FOC	B-site
of	O
EncFtnsH	B-protein
and	O
Pseudo	B-species
-	I-species
nitzschia	I-species
multiseries	I-species
ferritin	B-protein
(	O
PmFtn	B-protein
).	O

The	O
metal	B-site
binding	I-site
site	I-site
residues	O
from	O
two	O
EncFtnsH	B-protein
chains	O
are	O
shown	O
in	O
green	O
and	O
blue	O
,	O
while	O
the	O
PmFtn	B-protein
is	O
shown	O
in	O
orange	O
.	O

Fe2	B-chemical
+	I-chemical
in	O
the	O
FOC	B-site
is	O
shown	O
as	O
orange	O
spheres	O
and	O
Ca2	B-chemical
+	I-chemical
in	O
EncFtnsH	B-protein
is	O
shown	O
as	O
a	O
grey	O
sphere	O
.	O

The	O
two	O
-	O
fold	O
symmetry	O
axis	O
of	O
the	O
EncFtn	B-protein
FOC	B-site
is	O
shown	O
with	O
a	O
grey	O
arrow	O
(	O
B	O
)	O
Cross	O
-	O
section	O
surface	O
view	O
of	O
quaternary	O
structure	O
of	O
EncFtnsH	B-protein
and	O
PmFtn	B-protein
as	O
aligned	O
in	O
(	O
A	O
)	O
(	O
dashed	O
black	O
box	O
).	O

The	O
central	B-site
channel	I-site
of	O
EncFtnsH	B-protein
is	O
spatially	O
equivalent	O
to	O
the	O
outer	O
surface	O
of	O
ferritin	B-protein_type
and	O
its	O
outer	O
surface	O
corresponds	O
to	O
the	O
mineralization	B-site
surface	I-site
within	O
ferritin	B-protein_type
.	O

Comparison	B-experimental_method
of	O
the	O
symmetric	O
metal	B-site
ion	I-site
binding	I-site
site	I-site
of	O
EncFtnsH	B-protein
and	O
the	O
ferritin	B-protein_type
FOC	B-site
.	O

(	O
A	O
)	O
Structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
FOC	B-site
residues	O
in	O
a	O
dimer	B-oligomeric_state
of	O
EncFtnsH	B-protein
(	O
green	O
/	O
blue	O
)	O
with	O
a	O
monomer	B-oligomeric_state
of	O
Pseudo	B-species
-	I-species
nitzschia	I-species
multiseries	I-species
ferritin	B-protein
(	O
PmFtn	B-protein
)	O
(	O
PDBID	O
:	O
4ITW	O
)	O
(	O
orange	O
).	O

Iron	B-chemical
ions	O
are	O
shown	O
as	O
orange	O
spheres	O
and	O
a	O
single	O
calcium	B-chemical
ion	O
as	O
a	O
grey	O
sphere	O
.	O

Residues	O
within	O
the	O
FOC	B-site
are	O
conserved	B-protein_state
between	O
EncFtn	B-protein
and	O
ferritin	B-protein_type
PmFtn	B-protein
,	O
with	O
the	O
exception	O
of	O
residues	O
in	O
the	O
position	O
equivalent	O
to	O
H65	B-residue_name_number
’	O
in	O
the	O
second	O
subunit	B-oligomeric_state
in	O
the	O
dimer	B-oligomeric_state
(	O
blue	O
).	O

The	O
site	O
in	O
EncFtn	B-protein
with	O
bound	B-protein_state
calcium	B-chemical
is	O
not	O
present	O
in	O
other	O
family	O
members	O
.	O

(	O
B	O
)	O
Secondary	O
structure	O
of	O
aligned	B-experimental_method
dimeric	B-oligomeric_state
EncFtnsH	B-protein
and	O
monomeric	B-oligomeric_state
ferritin	B-protein_type
highlighting	O
the	O
conserved	B-protein_state
four	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
.	O

EncFtnsH	B-protein
monomers	B-oligomeric_state
are	O
shown	O
in	O
green	O
and	O
blue	O
and	O
aligned	B-experimental_method
PmFtn	B-protein
monomer	B-oligomeric_state
in	O
orange	O
as	O
in	O
A	O
.	O
(	O
C	O
)	O
Cartoon	O
of	O
secondary	O
structure	O
elements	O
in	O
EncFtn	B-protein
dimer	B-oligomeric_state
and	O
ferritin	B-protein_type
.	O

In	O
the	O
dimer	B-oligomeric_state
of	O
EncFtn	B-protein
that	O
forms	O
the	O
FOC	B-site
,	O
the	O
C	O
-	O
terminus	O
of	O
the	O
first	O
monomer	B-oligomeric_state
(	O
green	O
)	O
and	O
N	O
-	O
terminus	O
of	O
the	O
second	O
monomer	B-oligomeric_state
(	O
blue	O
)	O
correspond	O
to	O
the	O
position	O
of	O
the	O
long	B-structure_element
linker	I-structure_element
between	O
α2	B-structure_element
and	O
α3	B-structure_element
in	O
ferritin	B-protein_type
PmFtn	B-protein
.	O

Structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
di	B-site
-	I-site
iron	I-site
binding	I-site
site	I-site
of	O
EncFtnsH	B-protein
to	O
the	O
FOC	B-site
of	O
Pseudo	B-species
-	I-species
nitzschia	I-species
multiseries	I-species
ferritin	B-protein_type
(	O
PmFtn	B-protein
,	O
PDB	O
ID	O
:	O
4ITW	O
)	O
reveals	O
a	O
striking	O
similarity	O
between	O
the	O
metal	B-site
binding	I-site
sites	I-site
of	O
EncFtnsH	B-protein
and	O
the	O
classical	B-protein_state
ferritins	B-protein_type
(	O
Figure	O
6A	O
).	O

The	O
di	B-site
-	I-site
iron	I-site
site	I-site
of	O
EncFtnsH	B-protein
is	O
by	O
necessity	O
symmetrical	O
,	O
as	O
it	O
is	O
formed	O
through	O
a	O
dimer	B-site
interface	I-site
,	O
while	O
the	O
FOC	B-site
of	O
ferritin	B-protein_type
does	O
not	O
have	O
these	O
constraints	O
and	O
varies	O
in	O
different	O
species	O
at	O
a	O
position	O
equivalent	O
to	O
His65	B-residue_name_number
of	O
the	O
second	O
EncFtn	B-protein
monomer	B-oligomeric_state
in	O
the	O
FOC	B-site
interface	I-site
(	O
His65	B-residue_name_number
’)	O
(	O
Figure	O
6A	O
).	O

Structural	B-experimental_method
superimposition	I-experimental_method
of	O
the	O
FOCs	B-site
of	O
ferritin	B-protein_type
and	O
EncFtn	B-protein
brings	O
the	O
four	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
of	O
the	O
ferritin	B-protein_type
fold	O
into	O
close	O
alignment	O
with	O
the	O
EncFtn	B-protein
dimer	B-oligomeric_state
,	O
showing	O
that	O
the	O
two	O
families	O
of	O
proteins	O
have	O
essentially	O
the	O
same	O
architecture	O
around	O
the	O
di	B-site
-	I-site
iron	I-site
center	I-site
(	O
Figure	O
6B	O
).	O

The	O
linker	B-structure_element
connecting	O
helices	B-structure_element
2	I-structure_element
and	I-structure_element
3	I-structure_element
of	O
ferritin	B-protein_type
is	O
congruent	O
with	O
the	O
start	O
of	O
the	O
C	O
-	O
terminal	O
helix	B-structure_element
of	O
one	O
EncFtn	B-protein
monomer	B-oligomeric_state
and	O
the	O
N	O
-	O
terminal	O
310	B-structure_element
helix	I-structure_element
of	O
the	O
second	O
monomer	B-oligomeric_state
(	O
Figure	O
6C	O
).	O

Mass	B-experimental_method
spectrometry	I-experimental_method
of	O
the	O
EncFtn	B-protein
assembly	O

Native	B-experimental_method
IM	I-experimental_method
-	I-experimental_method
MS	I-experimental_method
analysis	O
of	O
the	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
.	O

(	O
A	O
)	O
Mass	B-evidence
spectrum	I-evidence
of	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
acquired	O
from	O
100	O
mM	O
ammonium	O
acetate	O
pH	O
8	O
.	O
0	O
under	O
native	B-experimental_method
MS	I-experimental_method
conditions	O
.	O

The	O
charge	B-evidence
state	I-evidence
distribution	O
observed	O
is	O
bimodal	O
,	O
with	O
peaks	B-evidence
corresponding	O
to	O
the	O
6	O
+	O
to	O
15	O
+	O
charge	B-evidence
states	I-evidence
of	O
apo	B-protein_state
-	O
monomer	B-oligomeric_state
EncFtnsH	B-protein
(	O
neutral	O
average	O
mass	O
13	O
,	O
194	O
.	O
3	O
Da	O
).	O
(	O
B	O
)	O
The	O
arrival	B-evidence
time	I-evidence
distributions	I-evidence
(	O
ion	B-evidence
mobility	I-evidence
data	I-evidence
)	O
of	O
all	O
ions	O
in	O
the	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
charge	B-evidence
state	I-evidence
distribution	O
displayed	O
as	O
a	O
greyscale	O
heat	O
map	O
(	O
linear	O
intensity	O
scale	O
).	O
(	O
B	O
)	O
Right	O
,	O
the	O
arrival	B-evidence
time	I-evidence
distribution	I-evidence
of	O
the	O
6	O
+	O
(	O
orange	O
)	O
and	O
7	O
+	O
(	O
green	O
)	O
charge	B-evidence
state	I-evidence
(	O
dashed	O
colored	O
‐	O
box	O
)	O
has	O
been	O
extracted	O
and	O
plotted	O
;	O
The	O
arrival	B-evidence
time	I-evidence
distributions	I-evidence
for	O
these	O
ion	O
is	O
shown	O
(	O
ms	O
),	O
along	O
with	O
the	O
calibrated	O
collision	B-evidence
cross	I-evidence
section	I-evidence
,	O
Ω	B-evidence
(	O
nm2	O
).	O
(	O
C	O
)	O
The	O
collision	B-evidence
cross	I-evidence
section	I-evidence
of	O
a	O
single	O
monomer	B-oligomeric_state
unit	O
from	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
Fe	B-protein_state
-	I-protein_state
loaded	I-protein_state
EncFtnsH	B-protein
decamer	B-oligomeric_state
was	O
calculated	O
to	O
be	O
15	O
.	O
8	O
nm2	O
using	O
IMPACT	O
v	O
.	O
0	O
.	O
9	O
.	O
1	O
.	O

The	O
+	O
8	O
to	O
+	O
15	O
protein	O
charge	B-evidence
states	I-evidence
have	O
observed	O
CCS	B-evidence
between	O
20	O
–	O
26	O
nm2	O
,	O
which	O
is	O
significantly	O
higher	O
than	O
the	O
calculated	O
CCS	B-evidence
for	O
an	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
taken	O
from	O
the	O
decameric	B-oligomeric_state
assembly	O
crystal	B-evidence
structure	I-evidence
(	O
15	O
.	O
8	O
nm2	O
).	O

The	O
mobility	B-evidence
of	O
the	O
+	O
7	O
charge	B-evidence
state	I-evidence
displays	O
broad	O
drift	B-evidence
-	I-evidence
time	I-evidence
distribution	I-evidence
with	O
maxima	O
consistent	O
with	O
CCS	B-evidence
of	O
15	O
.	O
9	O
and	O
17	O
.	O
9	O
nm2	O
.	O

Finally	O
,	O
the	O
6	O
+	O
charge	B-evidence
state	I-evidence
of	O
EncFtnsH	B-protein
has	O
mobility	B-evidence
consistent	O
with	O
a	O
CCS	B-evidence
of	O
12	O
.	O
3	O
nm2	O
,	O
indicating	O
a	O
more	O
compact	B-protein_state
/	O
collapsed	B-protein_state
structure	O
.	O

It	O
is	O
clear	O
from	O
this	O
data	O
that	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
exists	O
in	O
several	O
gas	O
phase	O
conformations	O
.	O

The	O
range	O
of	O
charge	B-evidence
states	I-evidence
occupied	O
by	O
the	O
protein	O
(	O
6	O
+	O
to	O
15	O
+)	O
and	O
the	O
range	O
of	O
CCS	B-evidence
in	O
which	O
the	O
protein	O
is	O
observed	O
(	O
12	O
.	O
3	O
nm2	O
–	O
26	O
nm2	O
)	O
are	O
both	O
large	O
.	O

In	O
addition	O
,	O
many	O
of	O
the	O
charge	B-evidence
states	I-evidence
observed	O
have	O
higher	O
charge	O
than	O
the	O
theoretical	O
maximal	O
charge	O
on	O
spherical	O
globular	B-protein_state
protein	O
,	O
as	O
determined	O
by	O
the	O
De	B-experimental_method
La	I-experimental_method
Mora	I-experimental_method
relationship	I-experimental_method
(	O
ZR	B-evidence
=	O
0	O
.	O
0778m	O
;	O
for	O
the	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
ZR	B-evidence
=	O
8	O
.	O
9	O
)	O
Fernandez	O
.	O

As	O
described	O
by	O
Beveridge	O
et	O
al	O
.,	O
all	O
these	O
factors	O
are	O
indicative	O
of	O
a	O
disordered	B-protein_state
protein	O
.	O

Gas	O
-	O
phase	O
disassembly	O
of	O
the	O
holo	B-protein_state
-	O
EncFtnsH	B-protein
decameric	B-oligomeric_state
assembly	O
.	O

The	O
entire	O
charge	B-evidence
state	I-evidence
distribution	O
of	O
the	O
Fe	B-protein_state
-	I-protein_state
loaded	I-protein_state
holo	B-protein_state
-	O
EncFtnsH	B-protein
assembly	O
(	O
green	O
circles	O
)	O
was	O
subject	O
to	O
collisional	B-experimental_method
-	I-experimental_method
induced	I-experimental_method
dissociation	I-experimental_method
(	O
CID	B-experimental_method
)	O
by	O
increasing	O
the	O
source	O
cone	O
voltage	O
to	O
200	O
V	O
and	O
the	O
trap	O
voltage	O
to	O
50	O
V	O
.	O
The	O
resulting	O
CID	B-experimental_method
mass	B-evidence
spectrum	I-evidence
(	O
A	O
)	O
revealed	O
that	O
dissociation	O
of	O
the	O
holo	B-protein_state
-	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
primarily	O
occurred	O
via	O
ejection	O
of	O
a	O
highly	O
charged	O
monomer	B-oligomeric_state
(	O
blue	O
circles	O
),	O
leaving	O
the	O
‘	O
stripped	B-protein_state
’	O
complex	O
(	O
a	O
9mer	B-oligomeric_state
;	O
118	O
.	O
7	O
kDa	O
;	O
yellow	O
circles	O
).	O

The	O
mass	O
of	O
the	O
ejected	O
-	O
monomer	B-oligomeric_state
is	O
consistent	O
with	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
(	O
13	O
.	O
2	O
kDa	O
),	O
suggesting	O
unfolding	O
of	O
the	O
monomer	B-oligomeric_state
(	O
and	O
loss	B-protein_state
of	I-protein_state
Fe	B-chemical
)	O
occurs	O
during	O
ejection	O
from	O
the	O
complex	O
.	O

This	O
observation	O
of	O
asymmetric	O
charge	O
partitioning	O
of	O
the	O
sub	O
-	O
complexes	O
with	O
respect	O
to	O
the	O
mass	O
of	O
the	O
complex	O
is	O
consistent	O
with	O
the	O
'	O
typical	O
'	O
pathway	O
of	O
dissociation	O
of	O
protein	O
assemblies	O
by	O
CID	B-experimental_method
,	O
as	O
described	O
by	O
.	O

In	O
addition	O
,	O
a	O
third	O
,	O
lower	O
abundance	O
,	O
charge	B-evidence
state	I-evidence
distribution	O
is	O
observed	O
which	O
overlaps	O
the	O
EncFtn	B-protein
ejected	O
monomer	B-oligomeric_state
charge	B-evidence
state	I-evidence
distribution	O
;	O
this	O
region	O
of	O
the	O
spectrum	O
is	O
highlighted	O
in	O
(	O
B	O
).	O

This	O
distribution	O
is	O
consistent	O
with	O
an	O
ejected	O
EncFtnsH	B-protein
dimer	B-oligomeric_state
(	O
orange	O
circles	O
).	O

Interestingly	O
,	O
closer	O
analysis	O
of	O
the	O
individual	O
charge	B-evidence
state	I-evidence
of	O
this	O
dimeric	B-oligomeric_state
CID	B-experimental_method
product	O
shows	O
that	O
this	O
sub	O
-	O
complex	O
exists	O
in	O
three	O
forms	O
–	O
displaying	O
mass	O
consistent	O
with	O
an	O
EncFtnsH	B-protein
dimer	B-oligomeric_state
binding	O
0	O
,	O
1	O
,	O
and	O
2	O
Fe	B-chemical
ions	O
.	O

This	O
is	O
highlighted	O
in	O
(	O
C	O
),	O
where	O
the	O
15	O
+	O
charge	B-evidence
state	I-evidence
of	O
the	O
EncFtnsH	B-protein
dimer	B-oligomeric_state
is	O
shown	O
;	O
3	O
peaks	B-evidence
are	O
observed	O
with	O
m	O
/	O
z	O
1760	O
.	O
5	O
,	O
1763	O
.	O
8	O
,	O
and	O
1767	O
.	O
0	O
Th	O
–	O
the	O
lowest	O
peak	O
corresponds	O
to	O
neutral	O
masses	O
of	O
26392	O
.	O
5	O
Da	O
[	O
predicted	O
EncFtnsH	B-protein
dimer	B-oligomeric_state
,	O
(	O
C572H884N172O185S2	O
)	O
2	O
;	O
26388	O
.	O
6	O
Da	O
].	O

The	O
two	O
further	O
peaks	B-evidence
have	O
a	O
delta	O
-	O
mass	O
of	O
~+	O
50	O
Da	O
,	O
consistent	O
with	O
Fe	B-chemical
binding	O
.	O

We	O
interpret	O
these	O
observations	O
as	O
partial	O
‘	O
atypical	O
’	O
CID	B-experimental_method
fragmentation	O
of	O
the	O
decameric	B-oligomeric_state
complex	O
–	O
i	O
.	O
e	O
.	O
fragmentation	O
of	O
the	O
initial	O
complex	O
with	O
retention	O
of	O
subunit	O
and	O
ligand	O
interactions	O
.	O

We	O
postulate	O
the	O
high	O
stability	O
of	O
this	O
iron	B-protein_state
-	I-protein_state
bound	I-protein_state
dimer	B-oligomeric_state
sub	O
-	O
complex	O
is	O
due	O
to	O
the	O
metal	B-chemical
coordination	B-bond_interaction
at	O
the	O
dimer	B-site
interface	I-site
,	O
increasing	O
the	O
strength	O
of	O
the	O
dimer	B-site
interface	I-site
.	O

Taken	O
together	O
,	O
these	O
observations	O
support	O
our	O
findings	O
that	O
the	O
topology	O
of	O
the	O
decameric	B-oligomeric_state
EncFtnsH	B-protein
assembly	O
is	O
arranged	O
as	O
a	O
pentamer	B-oligomeric_state
of	O
dimers	B-oligomeric_state
,	O
with	O
two	O
Fe	B-chemical
ions	O
at	O
each	O
dimer	B-site
interface	I-site
.	O

Native	B-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
and	O
ion	B-experimental_method
mobility	I-experimental_method
analysis	I-experimental_method
of	O
iron	B-chemical
loading	O
in	O
EncFtnsH	B-protein
.	O

All	O
spectra	B-evidence
were	O
acquired	O
in	O
100	O
mM	O
ammonium	O
acetate	B-chemical
,	O
pH	O
8	O
.	O
0	O
with	O
a	O
protein	O
concentration	O
of	O
5	O
µM	O
.	O
(	O
A	O
)	O
Native	B-experimental_method
nanoelectrospray	I-experimental_method
ionization	I-experimental_method
(	O
nESI	B-experimental_method
)	O
mass	B-experimental_method
spectrometry	I-experimental_method
of	O
EncFtnsH	B-protein
at	O
varying	O
iron	B-chemical
concentrations	O
.	O

A1	O
,	O
nESI	B-experimental_method
spectrum	B-evidence
of	O
iron	B-protein_state
-	I-protein_state
free	I-protein_state
EncFtnsH	B-protein
displays	O
a	O
charge	B-evidence
state	I-evidence
distribution	O
consistent	O
with	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
(	O
blue	O
circles	O
,	O
13	O
,	O
194	O
Da	O
).	O

Addition	O
of	O
100	O
µM	O
(	O
A2	O
)	O
and	O
300	O
µM	O
(	O
A3	O
)	O
Fe2	B-chemical
+	I-chemical
results	O
in	O
the	O
appearance	O
of	O
a	O
second	O
higher	O
molecular	B-evidence
weight	I-evidence
charge	B-evidence
state	I-evidence
distribution	O
consistent	O
with	O
a	O
decameric	B-oligomeric_state
assembly	O
of	O
EncFtnsH	B-protein
(	O
green	O
circles	O
,	O
132	O
.	O
6	O
kDa	O
).	O

(	O
B	O
)	O
Ion	B-experimental_method
mobility	I-experimental_method
(	I-experimental_method
IM	I-experimental_method
)-	I-experimental_method
MS	I-experimental_method
of	O
the	O
iron	B-protein_state
-	I-protein_state
bound	I-protein_state
holo	B-protein_state
-	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
.	O

Top	O
,	O
Peaks	B-evidence
corresponding	O
to	O
the	O
22	O
+	O
to	O
26	O
+	O
charge	B-evidence
states	I-evidence
of	O
a	O
homo	B-oligomeric_state
-	I-oligomeric_state
decameric	I-oligomeric_state
assembly	O
of	O
EncFtnsH	B-protein
are	O
observed	O
(	O
132	O
.	O
6	O
kDa	O
).	O

Top	O
Insert	O
,	O
Analysis	O
of	O
the	O
24	O
+	O
charge	B-evidence
state	I-evidence
of	O
the	O
assembly	O
at	O
m	O
/	O
z	O
5528	O
.	O
2	O
Th	O
.	O

The	O
theoretical	O
average	O
m	O
/	O
z	O
of	O
the	O
24	O
+	O
charge	B-evidence
state	I-evidence
with	O
no	O
additional	O
metals	O
bound	O
is	O
marked	O
by	O
a	O
red	O
line	O
(	O
5498	O
.	O
7	O
Th	O
);	O
the	O
observed	O
m	O
/	O
z	O
of	O
the	O
24	O
+	O
charge	B-evidence
state	I-evidence
indicates	O
that	O
the	O
EncFtnsH	B-protein
assembly	O
binds	O
between	O
10	O
(	O
green	O
line	O
,	O
5521	O
.	O
1	O
Th	O
)	O
and	O
15	O
Fe	B-chemical
ions	O
(	O
blue	O
line	O
,	O
5532	O
.	O
4	O
Th	O
)	O
per	O
decamer	B-oligomeric_state
.	O

Bottom	O
,	O
The	O
arrival	B-evidence
time	I-evidence
distributions	I-evidence
(	O
ion	B-evidence
mobility	I-evidence
data	I-evidence
)	O
of	O
all	O
ions	O
in	O
the	O
EncFtnsH	B-protein
charge	B-evidence
state	I-evidence
distribution	O
displayed	O
as	O
a	O
greyscale	O
heat	O
map	O
(	O
linear	O
intensity	O
scale	O
).	O

Bottom	O
right	O
,	O
The	O
arrival	B-evidence
time	I-evidence
distribution	I-evidence
of	O
the	O
24	O
+	O
charge	B-evidence
state	I-evidence
(	O
dashed	O
blue	O
box	O
)	O
has	O
been	O
extracted	O
and	O
plotted	O
.	O

The	O
drift	B-evidence
time	I-evidence
for	O
this	O
ion	O
is	O
shown	O
(	O
ms	O
),	O
along	O
with	O
the	O
calibrated	O
collision	B-evidence
cross	I-evidence
section	I-evidence
(	O
CCS	B-evidence
),	O
Ω	B-evidence
(	O
nm2	O
).	O

In	O
order	O
to	O
confirm	O
the	O
assignment	O
of	O
the	O
oligomeric	O
state	O
of	O
EncFtnsH	B-protein
and	O
investigate	O
further	O
the	O
Fe2	B-chemical
+-	I-chemical
dependent	O
assembly	O
,	O
we	O
used	O
native	B-experimental_method
nano	I-experimental_method
-	I-experimental_method
electrospray	I-experimental_method
ionization	I-experimental_method
(	O
nESI	B-experimental_method
)	O
and	O
ion	B-experimental_method
-	I-experimental_method
mobility	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
(	O
IM	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
).	O

As	O
described	O
above	O
,	O
by	O
recombinant	B-experimental_method
production	I-experimental_method
of	O
EncFtnsH	B-protein
in	O
minimal	O
media	O
we	O
were	O
able	O
to	O
limit	O
the	O
bioavailability	O
of	O
iron	B-chemical
.	O

Native	B-experimental_method
MS	I-experimental_method
analysis	O
of	O
EncFtnsH	B-protein
produced	O
in	O
this	O
way	O
displayed	O
a	O
charge	B-evidence
state	I-evidence
distribution	O
consistent	O
with	O
an	O
EncFtnsH	B-protein
monomer	B-oligomeric_state
(	O
blue	O
circles	O
,	O
Figure	O
7A1	O
)	O
with	O
an	O
average	O
neutral	O
mass	O
of	O
13	O
,	O
194	O
Da	O
,	O
in	O
agreement	O
with	O
the	O
predicted	O
mass	O
of	O
the	O
EncFtnsH	B-protein
protein	O
(	O
13	O
,	O
194	O
.	O
53	O
Da	O
).	O

Titration	B-experimental_method
with	O
Fe2	B-chemical
+	I-chemical
directly	O
before	O
native	B-experimental_method
MS	I-experimental_method
analysis	O
resulted	O
in	O
the	O
appearance	O
of	O
a	O
new	O
charge	B-evidence
state	I-evidence
distribution	O
,	O
consistent	O
with	O
an	O
EncFtnsH	B-protein
decameric	B-oligomeric_state
assembly	O
(+	O
22	O
to	O
+	O
26	O
;	O
132	O
.	O
65	O
kDa	O
)	O
(	O
Figure	O
7A2	O
/	O
3	O
).	O

After	O
instrument	O
optimization	O
,	O
the	O
mass	O
resolving	O
power	O
achieved	O
was	O
sufficient	O
to	O
assign	O
iron	B-chemical
-	O
loading	O
in	O
the	O
complex	O
to	O
between	O
10	O
and	O
15	O
Fe	B-chemical
ions	O
per	O
decamer	B-oligomeric_state
(	O
Figure	O
7B	O
,	O
inset	O
top	O
right	O
),	O
consistent	O
with	O
the	O
presence	B-protein_state
of	I-protein_state
10	O
irons	B-chemical
in	O
the	O
FOC	B-site
and	O
the	O
coordination	B-bond_interaction
of	O
iron	B-chemical
in	O
the	O
Glu31	B-site
/	I-site
34	I-site
-	I-site
site	I-site
occupied	O
by	O
calcium	B-chemical
in	O
the	O
crystal	B-evidence
structure	I-evidence
(	O
Δmass	B-evidence
observed	O
~	O
0	O
.	O
67	O
kDa	O
).	O

MS	B-experimental_method
analysis	O
of	O
EncFtnsH	B-protein
after	O
addition	O
of	O
further	O
Fe2	B-chemical
+	I-chemical
did	O
not	O
result	O
in	O
iron	B-chemical
loading	O
above	O
this	O
stoichiometry	O
.	O

Therefore	O
,	O
the	O
extent	O
of	O
iron	B-chemical
binding	O
seen	O
is	O
limited	O
to	O
the	O
FOC	B-site
and	O
Glu31	B-site
/	I-site
34	I-site
secondary	I-site
metal	I-site
binding	I-site
site	I-site
.	O

These	O
data	O
suggest	O
that	O
the	O
decameric	B-oligomeric_state
assembly	O
of	O
EncFtnsH	B-protein
does	O
not	O
accrue	O
iron	B-chemical
in	O
the	O
same	O
manner	O
as	O
classical	B-protein_state
ferritin	B-protein_type
,	O
which	O
is	O
able	O
to	O
sequester	O
around	O
4500	O
iron	B-chemical
ions	O
within	O
its	O
nanocage	B-complex_assembly
.	O

Ion	B-experimental_method
mobility	I-experimental_method
analysis	I-experimental_method
of	O
the	O
EncFtnsH	B-protein
decameric	B-oligomeric_state
assembly	O
,	O
collected	O
with	O
minimal	O
collisional	O
activation	O
,	O
suggested	O
that	O
it	O
consists	O
of	O
a	O
single	O
conformation	O
with	O
a	O
collision	B-evidence
cross	I-evidence
section	I-evidence
(	O
CCS	B-evidence
)	O
of	O
58	O
.	O
2	O
nm2	O
(	O
Figure	O
7B	O
).	O

This	O
observation	O
is	O
in	O
agreement	O
with	O
the	O
calculated	O
CCS	B-evidence
of	O
58	O
.	O
7	O
nm2derived	O
from	O
our	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
.	O

By	O
contrast	O
,	O
IM	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
measurements	O
of	O
the	O
monomeric	B-oligomeric_state
EncFtnsH	B-protein
at	O
pH	B-protein_state
8	I-protein_state
.	I-protein_state
0	I-protein_state
under	O
the	O
same	O
instrumental	O
conditions	O
revealed	O
that	O
the	O
metal	B-protein_state
-	I-protein_state
free	I-protein_state
protein	B-protein
monomer	B-oligomeric_state
exists	O
in	O
a	O
wide	O
range	O
of	O
charge	B-evidence
states	I-evidence
(+	O
6	O
to	O
+	O
16	O
)	O
and	O
adopts	O
many	O
conformations	O
in	O
the	O
gas	O
phase	O
with	O
collision	O
cross	O
sections	O
ranging	O
from	O
12	O
nm2	O
to	O
26	O
nm2	O
(	O
Figure	O
7	O
—	O
figure	O
supplement	O
1	O
).	O

Thus	O
,	O
IM	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
studies	O
highlight	O
that	O
higher	O
order	O
structure	O
in	O
EncFtnsH	B-protein
is	O
mediated	O
/	O
stabilized	O
by	O
metal	O
binding	O
,	O
an	O
observation	O
that	O
is	O
in	O
agreement	O
with	O
our	O
solution	O
studies	O
.	O

Taken	O
together	O
,	O
these	O
results	O
suggest	O
that	O
di	O
-	O
iron	B-chemical
binding	O
,	O
forming	O
the	O
FOC	B-site
in	O
EncFtnsH	B-protein
,	O
is	O
required	O
to	O
stabilize	O
the	O
4	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
dimer	B-site
interface	I-site
,	O
essentially	O
reconstructing	O
the	O
classical	B-protein_state
ferritin	B-protein_type
-	O
like	O
fold	O
;	O
once	O
stabilized	O
,	O
these	O
dimers	B-oligomeric_state
readily	O
associate	O
as	O
pentamers	O
,	O
and	O
the	O
overall	O
assembly	O
adopts	O
the	O
decameric	B-oligomeric_state
ring	O
arrangement	O
observed	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
.	O

We	O
subsequently	O
performed	O
gas	O
phase	O
disassembly	O
of	O
the	O
decameric	B-oligomeric_state
EncFtnsH	B-protein
using	O
collision	B-experimental_method
-	I-experimental_method
induced	I-experimental_method
dissociation	I-experimental_method
(	O
CID	B-experimental_method
)	O
tandem	B-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
.	O

Under	O
the	O
correct	O
CID	B-experimental_method
conditions	O
,	O
protein	O
assemblies	O
can	O
dissociate	O
with	O
retention	O
of	O
subunit	O
and	O
ligand	O
interactions	O
,	O
and	O
thus	O
provide	O
structurally	O
-	O
informative	O
evidence	O
as	O
to	O
the	O
topology	O
of	O
the	O
original	O
assembly	O
;	O
this	O
has	O
been	O
termed	O
‘	O
atypical	O
’	O
dissociation	O
.	O

For	O
EncFtnsH	B-protein
,	O
this	O
atypical	O
dissociation	O
pathway	O
was	O
clearly	O
evident	O
;	O
CID	B-experimental_method
of	O
the	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
resulted	O
in	O
the	O
appearance	O
of	O
a	O
dimeric	B-oligomeric_state
EncFtnsH	B-protein
subcomplex	O
containing	O
0	O
,	O
1	O
,	O
or	O
2	O
iron	B-chemical
ions	O
(	O
Figure	O
7	O
—	O
figure	O
supplement	O
2	O
).	O

In	O
light	O
of	O
the	O
crystal	B-evidence
structure	I-evidence
,	O
this	O
observation	O
can	O
be	O
rationalized	O
as	O
dissociation	O
of	O
the	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
by	O
disruption	O
of	O
the	O
non	B-site
-	I-site
FOC	I-site
interface	I-site
with	O
at	O
least	O
partial	O
retention	O
of	O
the	O
FOC	B-site
interface	I-site
and	O
the	O
FOC	B-site
-	O
Fe	B-chemical
.	O

Thus	O
,	O
this	O
observation	O
supports	O
our	O
crystallographic	O
assignment	O
of	O
the	O
overall	O
topology	O
of	O
the	O
EncFtnsH	B-protein
assembly	O
as	O
a	O
pentameric	B-oligomeric_state
assembly	O
of	O
dimers	B-oligomeric_state
with	O
two	O
iron	B-chemical
ions	O
located	O
at	O
the	O
FOC	B-site
dimer	I-site
interface	I-site
.	O

In	O
addition	O
,	O
this	O
analysis	O
provides	O
evidence	O
that	O
the	O
overall	O
architecture	O
of	O
the	O
complex	O
is	O
consistent	O
in	O
the	O
crystal	B-evidence
,	O
solution	O
and	O
gas	O
phases	O
.	O

Ferroxidase	B-protein_type
activity	O

TEM	B-experimental_method
visualization	O
of	O
iron	B-protein_state
-	I-protein_state
loaded	I-protein_state
bacterial	B-taxonomy_domain
nanocompartments	B-complex_assembly
and	O
ferritin	B-protein_type
.	O

Decameric	B-oligomeric_state
EncFtnsH	B-protein
,	O
encapsulin	B-protein
,	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
and	O
apoferritin	B-protein_state
,	O
at	O
8	O
.	O
5	O
µM	O
,	O
were	O
mixed	O
with	O
147	O
µM	O
,	O
1	O
mM	O
,	O
1	O
mM	O
and	O
215	O
µM	O
acidic	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
,	O
respectively	O
.	O

Protein	O
mixtures	O
were	O
incubated	O
at	O
room	O
temperature	O
for	O
1	O
hr	O
prior	O
to	O
TEM	B-experimental_method
analysis	O
with	O
or	O
without	O
uranyl	B-chemical
acetate	I-chemical
stain	O
.	O

(	O
A	O
–	O
D	O
)	O
Unstained	O
EncFtnsH	B-protein
,	O
encapsulin	B-protein
,	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
,	O
apoferritin	B-protein_state
loaded	B-protein_state
with	I-protein_state
Fe2	B-chemical
+,	I-chemical
respectively	O
,	O
with	O
35	O
,	O
000	O
x	O
magnification	O
and	O
scale	O
bars	O
indicate	O
100	O
nm	O
.	O
(	O
E	O
)	O
Protein	O
-	O
free	O
sample	O
as	O
a	O
control	O
.	O
(	O
F	O
–	O
I	O
)	O
Stained	B-experimental_method
EncFtnsH	B-protein
,	O
encapsulin	B-protein
,	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
,	O
apoferritin	B-protein_state
loaded	B-protein_state
with	I-protein_state
Fe2	B-chemical
+,	I-chemical
respectively	O
,	O
with	O
140	O
,	O
000	O
x	O
magnification	O
and	O
scale	O
bars	O
indicate	O
25	O
nm	O
.	O

Spectroscopic	O
evidence	O
for	O
the	O
ferroxidase	B-protein_type
activity	O
and	O
comparison	O
of	O
iron	B-chemical
loading	O
capacity	O
of	O
apoferritin	B-protein_state
,	O
EncFtnsH	B-protein
,	O
encapsulin	B-protein
,	O
and	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
.	O

(	O
A	O
)	O
Apoferritin	B-protein_state
(	O
10	O
μM	O
monomer	B-oligomeric_state
concentration	O
)	O
and	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
fractions	O
(	O
20	O
μM	O
monomer	B-oligomeric_state
concentration	O
,	O
10	O
μM	O
FOC	B-site
concentration	O
)	O
were	O
incubated	O
with	O
20	O
and	O
100	O
μM	O
iron	B-chemical
(	O
2	O
and	O
10	O
times	O
molar	O
equivalent	O
Fe2	B-chemical
+	I-chemical
per	O
FOC	B-site
)	O
and	O
progress	B-evidence
curves	I-evidence
of	O
the	O
oxidation	O
of	O
Fe2	B-chemical
+	I-chemical
to	O
Fe3	B-chemical
+	I-chemical
at	O
315	O
nm	O
were	O
recorded	O
in	O
a	O
spectrophotometer	O
.	O

The	O
background	O
oxidation	O
of	O
iron	B-chemical
at	O
20	O
and	O
100	O
μM	O
in	O
enzyme	O
-	O
free	O
controls	O
are	O
shown	O
for	O
reference	O
.	O
(	O
B	O
)	O
Encapsulin	B-protein
and	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
complexes	O
at	O
10	O
μM	O
asymmetric	O
unit	O
concentration	O
were	O
incubated	B-experimental_method
with	O
Fe2	B-chemical
+	I-chemical
at	O
20	O
and	O
100	O
μM	O
and	O
progress	B-evidence
curves	I-evidence
for	O
iron	B-chemical
oxidation	O
at	O
A315	O
were	O
measured	O
in	O
a	O
UV	B-experimental_method
/	I-experimental_method
visible	I-experimental_method
spectrophotometer	I-experimental_method
.	O

Enzyme	O
free	O
controls	O
for	O
background	O
oxidation	O
of	O
Fe2	B-chemical
+	I-chemical
are	O
shown	O
for	O
reference	O
.	O
(	O
C	O
)	O
Histogram	O
of	O
the	O
iron	B-chemical
loading	O
capacity	O
per	O
biological	O
assembly	O
of	O
EncFtnsH	B-protein
,	O
encapsulin	B-protein
,	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
and	O
apoferritin	B-protein_state
.	O

The	O
results	O
shown	O
are	O
for	O
three	O
technical	O
replicates	O
and	O
represent	O
the	O
optimal	O
iron	B-chemical
loading	O
by	O
the	O
complexes	O
after	O
three	O
hours	O
when	O
incubated	O
with	O
Fe2	B-chemical
+.	I-chemical

In	O
light	O
of	O
the	O
identification	O
of	O
an	O
iron	B-protein_state
-	I-protein_state
loaded	I-protein_state
FOC	B-site
in	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
EncFtn	B-protein
and	O
our	O
native	B-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
data	O
,	O
we	O
performed	O
ferroxidase	B-experimental_method
and	I-experimental_method
peroxidase	I-experimental_method
assays	I-experimental_method
to	O
demonstrate	O
the	O
catalytic	O
activity	O
of	O
this	O
protein	O
.	O

In	O
addition	O
,	O
we	O
also	O
assayed	O
equine	B-taxonomy_domain
apoferritin	B-protein_state
,	O
an	O
example	O
of	O
a	O
classical	B-protein_state
ferritin	B-protein_type
enzyme	O
,	O
as	O
a	O
positive	O
control	O
.	O

Unlike	O
the	O
Dps	B-protein_type
family	I-protein_type
of	O
ferritin	B-protein_type
-	I-protein_type
like	I-protein_type
proteins	I-protein_type
,	O
EncFtn	B-protein
showed	O
no	O
peroxidase	O
activity	O
when	O
assayed	O
with	O
the	O
substrate	O
ortho	B-chemical
-	I-chemical
phenylenediamine	I-chemical
.	O

The	O
ferroxidase	B-protein_type
activity	O
of	O
EncFtnsH	B-protein
was	O
measured	O
by	O
recording	O
the	O
progress	B-evidence
curve	I-evidence
of	O
Fe2	B-chemical
+	I-chemical
oxidation	O
to	O
Fe3	B-chemical
+	I-chemical
at	O
315	O
nm	O
after	O
addition	O
of	O
20	O
and	O
100	O
µM	O
Fe2	B-chemical
+	I-chemical
(	O
2	O
and	O
10	O
times	O
molar	O
ratio	O
Fe2	B-chemical
+/	I-chemical
FOC	B-site
).	O

In	O
both	O
experiments	O
the	O
rate	O
of	O
oxidation	O
was	O
faster	O
than	O
background	O
oxidation	O
of	O
Fe2	B-chemical
+	I-chemical
by	O
molecular	O
oxygen	B-chemical
,	O
and	O
was	O
highest	O
for	O
100	O
µM	O
Fe2	B-chemical
+	I-chemical
(	O
Figure	O
8A	O
).	O

These	O
data	O
show	O
that	O
recombinant	O
EncFtnsH	B-protein
acts	O
as	O
an	O
active	B-protein_state
ferroxidase	B-protein_type
enzyme	O
.	O

When	O
compared	O
to	O
apoferritin	B-protein_state
,	O
EncFtnsH	B-protein
oxidized	O
Fe2	B-chemical
+	I-chemical
at	O
a	O
slower	O
rate	O
and	O
the	O
reaction	O
did	O
not	O
run	O
to	O
completion	O
over	O
the	O
1800	O
s	O
of	O
the	O
experiment	O
.	O

Addition	O
of	O
higher	O
quantities	O
of	O
iron	B-chemical
resulted	O
in	O
the	O
formation	O
of	O
a	O
yellow	O
/	O
red	O
precipitate	O
at	O
the	O
end	O
of	O
the	O
reaction	O
.	O

We	O
also	O
performed	O
these	O
assays	O
on	O
purified	O
recombinant	O
encapsulin	B-protein
;	O
which	O
,	O
when	O
assayed	O
alone	O
,	O
did	O
not	O
display	O
ferroxidase	B-protein_type
activity	O
above	O
background	O
Fe2	B-chemical
+	I-chemical
oxidation	O
(	O
Figure	O
8B	O
).	O

In	O
contrast	O
,	O
complexes	O
of	O
the	O
full	B-protein_state
EncFtn	B-protein
encapsulin	B-protein
nanocompartment	B-complex_assembly
(	O
i	O
.	O
e	O
.	O
the	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
protein	O
complex	O
)	O
displayed	O
ferroxidase	B-protein_type
activity	O
comparable	O
to	O
apoferritin	B-protein_state
without	O
the	O
formation	O
of	O
precipitates	O
(	O
Figure	O
8B	O
).	O

We	O
attributed	O
the	O
precipitates	O
observed	O
in	O
the	O
EncFtnsH	B-protein
ferroxidase	B-experimental_method
assay	I-experimental_method
to	O
the	O
production	O
of	O
insoluble	O
Fe3	B-chemical
+	I-chemical
complexes	O
,	O
which	O
led	O
us	O
to	O
propose	O
that	O
EncFtn	B-protein
does	O
not	O
directly	O
store	O
Fe3	B-chemical
+	I-chemical
in	O
a	O
mineral	O
form	O
.	O

This	O
observation	O
agrees	O
with	O
native	B-experimental_method
MS	I-experimental_method
results	O
,	O
which	O
indicates	O
a	O
maximum	O
iron	B-chemical
loading	O
of	O
10	O
–	O
15	O
iron	B-chemical
ions	O
per	O
decameric	B-oligomeric_state
EncFtn	B-protein
;	O
and	O
the	O
structure	B-evidence
,	O
which	O
does	O
not	O
possess	O
the	O
enclosed	O
iron	B-site
-	I-site
storage	I-site
cavity	I-site
characteristic	O
of	O
classical	B-protein_state
ferritins	B-protein_type
and	O
Dps	B-protein_type
family	I-protein_type
proteins	I-protein_type
that	O
can	O
directly	O
accrue	O
mineralized	O
Fe3	B-chemical
+	I-chemical
within	O
their	O
nanocompartment	B-complex_assembly
structures	B-evidence
.	O

To	O
analyze	O
the	O
products	O
of	O
these	O
reactions	O
and	O
determine	O
whether	O
the	O
EncFtn	B-protein
and	O
encapsulin	B-protein
were	O
able	O
to	O
store	O
iron	B-chemical
in	O
a	O
mineral	O
form	O
,	O
we	O
performed	O
TEM	B-experimental_method
on	O
the	O
reaction	O
mixtures	O
from	O
the	O
ferroxidase	B-experimental_method
assay	I-experimental_method
.	O

The	O
EncFtnsH	B-protein
reaction	O
mixture	O
showed	O
the	O
formation	O
of	O
large	O
,	O
irregular	O
electron	O
-	O
dense	O
precipitates	O
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1A	O
).	O

A	O
similar	O
distribution	O
of	O
particles	O
was	O
observed	O
after	O
addition	O
of	O
Fe2	B-chemical
+	I-chemical
to	O
the	O
encapsulin	B-protein
protein	O
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1B	O
).	O

In	O
contrast	O
,	O
addition	O
of	O
Fe2	B-chemical
+	I-chemical
to	O
the	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartment	B-complex_assembly
resulted	O
in	O
small	O
,	O
highly	O
regular	O
,	O
electron	O
dense	O
particles	O
of	O
approximately	O
5	O
nm	O
in	O
diameter	O
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1C	O
);	O
we	O
interpret	O
these	O
observations	O
as	O
controlled	O
mineralization	O
of	O
iron	B-chemical
within	O
the	O
nanocompartment	B-complex_assembly
.	O

Addition	O
of	O
Fe2	B-chemical
+	I-chemical
to	O
apoferritin	B-protein_state
resulted	O
in	O
a	O
mixture	O
of	O
large	O
particles	O
and	O
small	O
(~	O
2	O
nm	O
)	O
particles	O
consistent	O
with	O
partial	O
mineralization	O
by	O
the	O
ferritin	B-protein_type
and	O
some	O
background	O
oxidation	O
of	O
the	O
iron	B-chemical
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1D	O
).	O

Negative	B-experimental_method
stain	I-experimental_method
TEM	I-experimental_method
of	O
these	O
samples	O
revealed	O
that	O
upon	O
addition	O
of	O
iron	B-chemical
,	O
the	O
EncFtnsH	B-protein
protein	O
showed	O
significant	O
aggregation	O
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1F	O
);	O
while	O
the	O
encapsulin	B-protein
,	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
system	O
,	O
and	O
apoferritin	B-protein_state
are	O
present	O
as	O
distinct	O
nanocompartments	B-complex_assembly
without	O
significant	O
protein	O
aggregation	O
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1G	O
–	O
I	O
).	O

Iron	B-chemical
storage	O
in	O
encapsulin	B-protein
nanocompartments	B-complex_assembly

The	O
results	O
of	O
the	O
ferroxidase	B-experimental_method
assay	I-experimental_method
and	O
micrographs	B-evidence
of	O
the	O
reaction	O
products	O
suggest	O
that	O
the	O
oxidation	O
and	O
mineralization	O
function	O
of	O
the	O
classical	B-protein_state
ferritins	B-protein_type
are	O
split	O
between	O
the	O
EncFtn	B-protein
and	O
encapsulin	B-protein
proteins	O
,	O
with	O
the	O
EncFtn	B-protein
acting	O
as	O
a	O
ferroxidase	B-protein_type
and	O
the	O
encapsulin	B-protein
shell	B-structure_element
providing	O
an	O
environment	O
and	O
template	O
for	O
iron	B-chemical
mineralization	O
and	O
storage	O
.	O

To	O
investigate	O
this	O
further	O
,	O
we	O
added	O
Fe2	B-chemical
+	I-chemical
at	O
various	O
concentrations	O
to	O
samples	O
of	O
apo	B-protein_state
-	O
ferritin	B-protein_type
,	O
EncFtn	B-protein
,	O
isolated	O
encapsulin	B-protein
,	O
and	O
the	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
protein	O
complex	O
,	O
and	O
subjected	O
these	O
samples	O
to	O
a	O
ferrozine	B-experimental_method
assay	I-experimental_method
to	O
quantify	O
the	O
amount	O
of	O
iron	B-chemical
associated	O
with	O
the	O
proteins	O
after	O
three	O
hours	O
of	O
incubation	O
.	O

The	O
maximum	O
iron	B-chemical
loading	O
capacity	O
of	O
these	O
systems	O
was	O
calculated	O
as	O
the	O
quantity	O
of	O
iron	B-chemical
per	O
biological	O
assembly	O
(	O
Figure	O
8C	O
).	O

In	O
this	O
assay	O
,	O
the	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
binds	O
a	O
maximum	O
of	O
around	O
48	O
iron	B-chemical
ions	O
before	O
excess	O
iron	B-chemical
induces	O
protein	O
precipitation	O
.	O

The	O
encapsulin	B-protein
shell	B-structure_element
protein	O
can	O
sequester	O
about	O
2200	O
iron	B-chemical
ions	O
before	O
significant	O
protein	O
loss	O
occurs	O
,	O
and	O
the	O
reconstituted	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartment	B-complex_assembly
sequestered	O
about	O
4150	O
iron	B-chemical
ions	O
.	O

This	O
latter	O
result	O
is	O
significantly	O
more	O
than	O
the	O
apoferritin	B-protein_state
used	O
in	O
our	O
assay	O
,	O
which	O
sequesters	O
approximately	O
570	O
iron	B-chemical
ions	O
in	O
this	O
assay	O
(	O
Figure	O
8C	O
,	O
Table	O
5	O
).	O

Consideration	O
of	O
the	O
functional	O
oligomeric	O
states	O
of	O
these	O
proteins	O
,	O
where	O
EncFtn	B-protein
is	O
a	O
decamer	B-oligomeric_state
and	O
encapsulin	B-protein
forms	O
an	O
icosahedral	B-protein_state
cage	B-complex_assembly
,	O
and	O
estimation	O
of	O
the	O
iron	B-chemical
loading	O
capacity	O
of	O
these	O
complexes	O
gives	O
insight	O
into	O
the	O
role	O
of	O
the	O
two	O
proteins	O
in	O
iron	B-chemical
storage	O
and	O
mineralization	O
.	O

EncFtn	B-protein
decamers	B-oligomeric_state
bind	O
up	O
to	O
48	O
iron	B-chemical
ions	O
(	O
Figure	O
8C	O
),	O
which	O
is	O
significantly	O
higher	O
than	O
the	O
stoichiometry	O
of	O
fifteen	O
metal	O
ions	O
visible	O
in	O
the	O
FOC	B-site
and	O
E31	B-site
/	I-site
34	I-site
-	I-site
site	I-site
of	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
and	O
our	O
MS	B-experimental_method
analysis	O
.	O

The	O
discrepancy	O
between	O
these	O
solution	B-experimental_method
measurements	I-experimental_method
and	O
our	O
MS	B-experimental_method
analysis	O
may	O
indicate	O
that	O
there	O
are	O
additional	O
metal	B-site
-	I-site
binding	I-site
sites	I-site
on	O
the	O
interior	O
channel	B-site
and	O
exterior	O
faces	O
of	O
the	O
protein	O
;	O
this	O
is	O
consistent	O
with	O
our	O
identification	O
of	O
a	O
number	O
of	O
weak	O
metal	B-site
-	I-site
binding	I-site
sites	I-site
at	O
the	O
surface	O
of	O
the	O
protein	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
(	O
Figure	O
5D	O
).	O

These	O
observations	O
are	O
consistent	O
with	O
hydrated	O
Fe2	B-chemical
+	I-chemical
ions	O
being	O
channeled	O
to	O
the	O
active	B-site
site	I-site
from	O
the	O
E31	B-site
/	I-site
34	I-site
-	I-site
site	I-site
and	O
the	O
subsequent	O
exit	O
of	O
Fe3	B-chemical
+	I-chemical
products	O
on	O
the	O
outer	O
surface	O
,	O
as	O
is	O
seen	O
in	O
other	O
ferritin	B-protein_type
family	O
proteins	O
.	O

While	O
the	O
isolated	O
encapsulin	B-protein
shell	B-structure_element
does	O
not	O
display	O
any	O
ferroxidase	B-protein_type
activity	O
,	O
it	O
binds	O
around	O
2200	O
iron	B-chemical
ions	O
in	O
our	O
assay	O
(	O
Table	O
5	O
).	O

This	O
implies	O
that	O
the	O
shell	B-structure_element
can	O
bind	O
a	O
significant	O
amount	O
of	O
iron	B-chemical
on	O
its	O
outer	O
and	O
inner	O
surfaces	O
.	O

While	O
the	O
maximum	O
reported	O
loading	O
capacity	O
of	O
classical	B-protein_state
ferritins	B-protein_type
is	O
approximately	O
4500	O
iron	B-chemical
ions	O
,	O
in	O
our	O
assay	O
system	O
we	O
were	O
only	O
able	O
to	O
load	O
apoferritin	B-protein_state
with	O
around	O
570	O
iron	B-chemical
ions	O
.	O

However	O
,	O
the	O
recombinant	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartment	B-complex_assembly
was	O
able	O
to	O
bind	O
over	O
4100	O
iron	B-chemical
ions	O
in	O
the	O
same	O
time	O
period	O
,	O
over	O
seven	O
times	O
the	O
amount	O
seen	O
for	O
the	O
apoferritin	B-protein_state
.	O

We	O
note	O
we	O
do	O
not	O
reach	O
the	O
experimental	O
maximum	O
iron	B-chemical
loading	O
for	O
apoferritin	B-protein_state
and	O
therefore	O
the	O
total	O
iron	B-chemical
-	O
loading	O
capacity	O
of	O
our	O
system	O
may	O
be	O
significantly	O
higher	O
than	O
in	O
this	O
experimental	O
system	O
.	O

Taken	O
together	O
,	O
our	O
data	O
show	O
that	O
EncFtn	B-protein
can	O
catalytically	O
oxidize	O
Fe2	B-chemical
+	I-chemical
to	O
Fe3	B-chemical
+;	I-chemical
however	O
,	O
iron	B-chemical
binding	O
in	O
EncFtn	B-protein
is	O
limited	O
to	O
the	O
FOC	B-site
and	O
several	O
surface	O
metal	B-site
binding	I-site
sites	I-site
.	O

In	O
contrast	O
,	O
the	O
encapsulin	B-protein
protein	O
displays	O
no	O
catalytic	O
activity	O
,	O
but	O
has	O
the	O
ability	O
to	O
bind	O
a	O
considerable	O
amount	O
of	O
iron	B-chemical
.	O

Finally	O
,	O
the	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartment	B-complex_assembly
complex	O
retains	O
the	O
catalytic	O
activity	O
of	O
EncFtn	B-protein
,	O
and	O
sequesters	O
iron	B-chemical
within	O
the	O
encapsulin	B-protein
shell	B-structure_element
at	O
a	O
higher	O
level	O
than	O
the	O
isolated	O
components	O
of	O
the	O
system	O
,	O
and	O
at	O
a	O
significantly	O
higher	O
level	O
than	O
the	O
classical	B-protein_state
ferritins	B-protein_type
.	O

Furthermore	O
,	O
our	O
recombinant	O
nanocompartments	B-complex_assembly
may	O
not	O
have	O
the	O
physiological	O
subunit	O
stoichiometry	O
,	O
and	O
the	O
iron	B-chemical
-	O
loading	O
capacity	O
of	O
native	B-protein_state
nanocompartments	B-complex_assembly
is	O
potentially	O
much	O
higher	O
than	O
the	O
level	O
we	O
have	O
observed	O
.	O

Mutagenesis	B-experimental_method
of	O
the	O
EncFtnsHferroxidase	B-protein
center	B-site

Purification	O
of	O
recombinant	O
R	B-species
.	I-species
rubrum	I-species
EncFtnsH	B-protein
FOC	B-site
mutants	B-protein_state
.	O

Single	O
mutants	B-protein_state
E32A	B-mutant
,	O
E62A	B-mutant
,	O
and	O
H65A	B-mutant
of	O
EncFtnsH	B-protein
produced	O
from	O
E	B-species
.	I-species
coli	I-species
BL21	I-species
(	I-species
DE3	I-species
)	I-species
cells	O
grown	O
in	O
MM	B-experimental_method
and	O
MM	B-experimental_method
supplemented	O
with	O
iron	B-chemical
were	O
subjected	O
to	O
Superdex	O
200	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
.	O

(	O
A	O
)	O
Gel	B-evidence
-	I-evidence
filtration	I-evidence
chromatogram	I-evidence
of	O
the	O
E32A	B-mutant
mutant	B-protein_state
form	O
of	O
EncFtnsH	B-protein
resulted	O
in	O
an	O
elution	B-evidence
profile	I-evidence
with	O
a	O
majority	O
of	O
the	O
protein	O
eluting	O
as	O
the	O
decameric	B-oligomeric_state
form	O
of	O
the	O
protein	O
and	O
a	O
small	O
proportion	O
of	O
monomer	B-oligomeric_state
.	O
(	O
B	O
)	O
Gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
chromatograhy	I-experimental_method
of	O
the	O
E62A	B-mutant
mutant	B-protein_state
form	O
of	O
EncFtnsH	B-protein
resulted	O
in	O
an	O
elution	B-evidence
profile	I-evidence
with	O
a	O
single	O
major	O
decameric	B-oligomeric_state
peak	O
.	O
(	O
C	O
)	O
Gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
chromatography	I-experimental_method
of	O
the	O
H65A	B-mutant
mutant	B-protein_state
form	O
of	O
EncFtnsH	B-protein
resulted	O
in	O
a	O
single	O
peak	O
corresponding	O
to	O
the	O
protein	O
monomer	B-oligomeric_state
.	O

To	O
investigate	O
the	O
structural	O
and	O
biochemical	O
role	O
played	O
by	O
the	O
metal	B-site
binding	I-site
residues	I-site
in	O
the	O
di	B-site
-	I-site
iron	I-site
FOC	I-site
of	O
EncFtnsH	B-protein
we	O
produced	O
alanine	B-experimental_method
mutations	I-experimental_method
in	O
each	O
of	O
these	O
residues	O
:	O
Glu32	B-residue_name_number
,	O
Glu62	B-residue_name_number
,	O
and	O
His65	B-residue_name_number
.	O

These	O
EncFtnsH	B-protein
mutants	B-protein_state
were	O
produced	O
in	O
E	B-species
.	I-species
coli	I-species
cells	O
grown	O
in	O
MM	B-experimental_method
,	O
both	O
in	O
the	O
absence	B-protein_state
and	O
presence	B-protein_state
of	I-protein_state
additional	O
iron	B-chemical
.	O

The	O
E32A	B-mutant
and	O
E62A	B-mutant
mutants	B-protein_state
eluted	O
from	O
SEC	B-experimental_method
at	O
a	O
volume	O
consistent	O
with	O
the	O
decameric	B-oligomeric_state
form	O
of	O
EncFtnsH	B-protein
,	O
with	O
a	O
small	O
proportion	O
of	O
monomer	B-oligomeric_state
;	O
the	O
H65A	B-mutant
mutant	B-protein_state
eluted	O
at	O
a	O
volume	O
consistent	O
with	O
the	O
monomeric	B-oligomeric_state
form	O
of	O
EncFtnsH	B-protein
(	O
Figure	O
9	O
).	O

For	O
all	O
of	O
the	O
mutants	B-protein_state
studied	O
,	O
no	O
change	O
in	O
oligomerization	O
state	O
was	O
apparent	O
upon	O
addition	O
of	O
Fe2	B-chemical
+	I-chemical
in	O
vitro	O
.	O

Native	B-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
of	O
EncFtnsH	B-protein
mutants	B-protein_state
.	O

All	O
spectra	B-evidence
were	O
acquired	O
in	O
100	O
mM	O
ammonium	O
acetate	B-chemical
,	O
pH	O
8	O
.	O
0	O
with	O
a	O
protein	O
concentration	O
of	O
5	O
µM	O
.	O
(	O
A	O
)	O
Wild	B-protein_state
-	I-protein_state
type	I-protein_state
EncFtnsH	B-protein
in	O
the	O
absence	B-protein_state
of	I-protein_state
iron	B-chemical
displays	O
a	O
charge	B-evidence
state	I-evidence
distribution	I-evidence
consistent	O
with	O
a	O
monomer	B-oligomeric_state
(	O
see	O
also	O
Figure	O
8	O
).	O
(	O
B	O
)	O
E32A	B-mutant
EncFtnsH	B-protein
displays	O
a	O
charge	B-evidence
states	I-evidence
consistent	O
with	O
a	O
decamer	B-oligomeric_state
(	O
green	O
circles	O
);	O
a	O
minor	O
species	O
,	O
consistent	O
with	O
the	O
monomer	B-oligomeric_state
of	O
E32A	B-mutant
mutant	B-protein_state
is	O
also	O
observed	O
(	O
blue	O
circles	O
).	O

(	O
C	O
)	O
E62A	B-mutant
EncFtnsH	B-protein
displays	O
charge	B-evidence
states	I-evidence
consistent	O
with	O
a	O
decamer	B-oligomeric_state
(	O
green	O
circles	O
).	O
(	O
D	O
)	O
H65A	B-mutant
EncFtnsH	B-protein
displays	O
charge	B-evidence
states	I-evidence
consistent	O
with	O
both	O
monomer	B-oligomeric_state
(	O
blue	O
circles	O
)	O
and	O
dimer	B-oligomeric_state
(	O
purple	O
circles	O
).	O

In	O
addition	O
to	O
SEC	B-experimental_method
studies	O
,	O
native	B-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
of	O
the	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
mutants	B-protein_state
was	O
performed	O
and	O
compared	O
with	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
apo	B-protein_state
-	O
EncFtnsH	B-protein
protein	O
(	O
Figure	O
10	O
).	O

As	O
described	O
above	O
,	O
the	O
apo	B-protein_state
-	O
EncFtnsH	B-protein
has	O
a	O
charge	B-evidence
state	I-evidence
distribution	O
consistent	O
with	O
an	O
unstructured	B-protein_state
monomer	B-oligomeric_state
,	O
and	O
decamer	B-oligomeric_state
formation	O
is	O
only	O
initiated	O
upon	O
addition	O
of	O
ferrous	O
iron	B-chemical
.	O

Both	O
the	O
E32A	B-mutant
mutant	B-protein_state
and	O
E62A	B-mutant
mutant	B-protein_state
displayed	O
charge	B-evidence
state	I-evidence
distributions	O
consistent	O
with	O
decamers	B-oligomeric_state
,	O
even	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
Fe2	B-chemical
+.	I-chemical

This	O
gas	O
-	O
phase	O
observation	O
is	O
consistent	O
with	O
SEC	B-experimental_method
measurements	O
,	O
which	O
indicate	O
both	O
of	O
these	O
variants	O
were	O
also	O
decamers	B-oligomeric_state
in	O
solution	O
.	O

Thus	O
it	O
seems	O
that	O
these	O
mutations	O
allow	O
the	O
decamer	B-oligomeric_state
to	O
form	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
iron	B-chemical
in	O
the	O
FOC	B-site
.	O

In	O
contrast	O
to	O
the	O
glutamic	B-residue_name
acid	I-residue_name
mutants	B-protein_state
,	O
MS	B-experimental_method
analysis	O
of	O
the	O
H65A	B-mutant
mutant	B-protein_state
is	O
similar	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
apo	B-protein_state
-	O
EncFtnsH	B-protein
and	O
is	O
present	O
as	O
a	O
monomer	B-oligomeric_state
;	O
interestingly	O
a	O
minor	O
population	O
of	O
dimeric	B-oligomeric_state
H65A	B-mutant
was	O
also	O
observed	O
.	O

We	O
propose	O
that	O
the	O
observed	O
differences	O
in	O
the	O
oligomerization	O
state	O
of	O
the	O
E32A	B-mutant
and	O
E62A	B-mutant
mutants	B-protein_state
compared	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
are	O
due	O
to	O
the	O
changes	O
in	O
the	O
electrostatic	O
environment	O
within	O
the	O
FOC	B-site
.	O

At	O
neutral	B-protein_state
pH	I-protein_state
the	O
glutamic	B-residue_name
acid	I-residue_name
residues	O
are	O
negatively	O
charged	O
,	O
while	O
the	O
histidine	B-residue_name
residues	O
are	O
predominantly	O
in	O
their	O
uncharged	O
state	O
.	O

In	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
EncFtnsH	B-protein
this	O
leads	O
to	O
electrostatic	O
repulsion	O
between	O
subunits	B-structure_element
in	O
the	O
absence	B-protein_state
of	I-protein_state
iron	B-chemical
.	O

Coordination	B-bond_interaction
of	O
Fe2	B-chemical
+	I-chemical
in	O
this	O
site	O
stabilizes	O
the	O
dimer	B-oligomeric_state
and	O
reconstitutes	O
the	O
active	B-protein_state
FOC	B-site
.	O

The	O
geometric	O
arrangement	O
of	O
Glu32	B-residue_name_number
and	O
Glu62	B-residue_name_number
in	O
the	O
FOC	B-site
explains	O
their	O
behavior	O
in	O
solution	O
and	O
the	O
gas	O
phase	O
,	O
where	O
they	O
both	O
favor	O
the	O
formation	O
of	O
decamers	B-oligomeric_state
due	O
to	O
the	O
loss	O
of	O
a	O
repulsive	O
negative	O
charge	O
.	O

The	O
FOC	B-site
in	O
the	O
H65A	B-mutant
mutant	B-protein_state
is	O
destabilized	O
through	O
the	O
loss	B-protein_state
of	I-protein_state
this	O
metal	B-site
coordinating	I-site
residue	I-site
and	O
potential	O
positive	O
charge	O
carrier	O
,	O
thus	O
favoring	O
the	O
monomer	B-oligomeric_state
in	O
solution	O
and	O
the	O
gas	O
phase	O
.	O

Data	B-evidence
collection	I-evidence
and	I-evidence
refinement	I-evidence
statistics	I-evidence
.	O

WT	B-protein_state
E32A	B-mutant
E62A	B-mutant
H65A	B-mutant
Data	O
collection	O
Wavelength	O
(	O
Å	O
)	O
1	O
.	O
74	O
1	O
.	O
73	O
1	O
.	O
73	O
1	O
.	O
74	O
Resolution	O
range	O
(	O
Å	O
)	O
49	O
.	O
63	O
-	O
2	O
.	O
06	O
(	O
2	O
.	O
10	O
-	O
2	O
.	O
06	O
)	O
48	O
.	O
84	O
-	O
2	O
.	O
59	O
(	O
2	O
.	O
683	O
-	O
2	O
.	O
59	O
)	O
48	O
.	O
87	O
-	O
2	O
.	O
21	O
(	O
2	O
.	O
29	O
-	O
2	O
.	O
21	O
)	O
48	O
.	O
86	O
-	O
2	O
.	O
97	O
(	O
3	O
.	O
08	O
-	O
2	O
.	O
97	O
)	O
Space	O
group	O
P	O
1	O
21	O
1	O
P	O
1	O
21	O
1	O
P	O
1	O
21	O
1	O
P	O
1	O
21	O
1	O
Unit	O
cell	O
(	O
Å	O
)	O
a	O
b	O
c	O
β	O
(°)	O
98	O
.	O
18	O
120	O
.	O
53	O
140	O
.	O
30	O
95	O
.	O
36	O
97	O
.	O
78	O
120	O
.	O
28	O
140	O
.	O
53	O
95	O
.	O
41	O
98	O
.	O
09	O
120	O
.	O
23	O
140	O
.	O
36	O
95	O
.	O
50	O
98	O
.	O
03	O
120	O
.	O
29	O
140	O
.	O
43	O
95	O
.	O
39	O
Total	O
reflections	O
1	O
,	O
264	O
,	O
922	O
(	O
41	O
,	O
360	O
)	O
405	O
,	O
488	O
(	O
36	O
,	O
186	O
)	O
1	O
,	O
069	O
,	O
345	O
(	O
95	O
,	O
716	O
)	O
323	O
,	O
853	O
(	O
32	O
,	O
120	O
)	O
Unique	O
reflections	O
197	O
,	O
873	O
(	O
8	O
,	O
766	O
)	O
100	O
,	O
067	O
(	O
9	O
,	O
735	O
)	O
162	O
,	O
379	O
(	O
15	O
,	O
817	O
)	O
66	O
,	O
658	O
(	O
6	O
,	O
553	O
)	O
Multiplicity	O
6	O
.	O
4	O
(	O
4	O
.	O
7	O
)	O
4	O
.	O
1	O
(	O
3	O
.	O
7	O
)	O
6	O
.	O
6	O
(	O
6	O
.	O
1	O
)	O
4	O
.	O
9	O
(	O
4	O
.	O
9	O
)	O
Anomalous	O
multiplicity	O
3	O
.	O
2	O
(	O
2	O
.	O
6	O
)	O
N	O
/	O
A	O
N	O
/	O
A	O
N	O
/	O
A	O
Completeness	O
(%)	O
99	O
.	O
2	O
(	O
88	O
.	O
6	O
)	O
99	O
.	O
0	O
(	O
97	O
.	O
0	O
)	O
100	O
(	O
97	O
.	O
0	O
)	O
100	O
(	O
99	O
.	O
0	O
)	O
Anomalous	O
completeness	O
(%)	O
96	O
.	O
7	O
(	O
77	O
.	O
2	O
)	O
N	O
/	O
A	O
N	O
/	O
A	O
N	O
/	O
A	O
Mean	O
I	O
/	O
sigma	O
(	O
I	O
)	O
10	O
.	O
6	O
(	O
1	O
.	O
60	O
)	O
8	O
.	O
46	O
(	O
1	O
.	O
79	O
)	O
13	O
.	O
74	O
(	O
1	O
.	O
80	O
)	O
8	O
.	O
09	O
(	O
1	O
.	O
74	O
)	O
Wilson	O
B	O
-	O
factor	O
26	O
.	O
98	O
40	O
.	O
10	O
33	O
.	O
97	O
52	O
.	O
20	O
Rmerge	O
0	O
.	O
123	O
(	O
0	O
.	O
790	O
)	O
0	O
.	O
171	O
(	O
0	O
.	O
792	O
)	O
0	O
.	O
0979	O
(	O
1	O
.	O
009	O
)	O
0	O
.	O
177	O
(	O
0	O
.	O
863	O
)	O
Rmeas	O
0	O
.	O
147	O
(	O
0	O
.	O
973	O
)	O
0	O
.	O
196	O
(	O
0	O
.	O
923	O
)	O
0	O
.	O
1064	O
(	O
1	O
.	O
107	O
)	O
0	O
.	O
199	O
(	O
0	O
.	O
966	O
)	O
CC1	O
/	O
2	O
0	O
.	O
995	O
(	O
0	O
.	O
469	O
)	O
0	O
.	O
985	O
(	O
0	O
.	O
557	O
)	O
0	O
.	O
998	O
(	O
0	O
.	O
642	O
)	O
0	O
.	O
989	O
(	O
0	O
.	O
627	O
)	O
CC	O
*	O
0	O
.	O
999	O
(	O
0	O
.	O
846	O
)	O
0	O
.	O
996	O
(	O
0	O
.	O
846	O
)	O
0	O
.	O
999	O
(	O
0	O
.	O
884	O
)	O
0	O
.	O
997	O
(	O
0	O
.	O
878	O
)	O
Image	O
DOI	O
10	O
.	O
7488	O
/	O
ds	O
/	O
1342	O
10	O
.	O
7488	O
/	O
ds	O
/	O
1419	O
10	O
.	O
7488	O
/	O
ds	O
/	O
1420	O
10	O
.	O
7488	O
/	O
ds	O
/	O
1421	O
Refinement	O
Rwork	O
0	O
.	O
171	O
(	O
0	O
.	O
318	O
)	O
0	O
.	O
183	O
(	O
0	O
.	O
288	O
)	O
0	O
.	O
165	O
(	O
0	O
.	O
299	O
)	O
0	O
.	O
186	O
(	O
0	O
.	O
273	O
)	O
Rfree	O
0	O
.	O
206	O
(	O
0	O
.	O
345	O
)	O
0	O
.	O
225	O
(	O
0351	O
)	O
0	O
.	O
216	O
(	O
0	O
.	O
364	O
)	O
0	O
.	O
237	O
(	O
0	O
.	O
325	O
)	O
Number	O
of	O
non	O
-	O
hydrogen	O
atoms	O
23	O
,	O
222	O
22	O
,	O
366	O
22	O
,	O
691	O
22	O
,	O
145	O
macromolecules	O
22	O
,	O
276	O
22	O
,	O
019	O
21	O
,	O
965	O
22	O
,	O
066	O
ligands	O
138	O
8	O
24	O
74	O
water	B-chemical
808	O
339	O
702	O
5	O
Protein	O
residues	O
2	O
,	O
703	O
2	O
,	O
686	O
2	O
,	O
675	O
2	O
,	O
700	O
RMS	O
(	O
bonds	O
)	O
(	O
Å	O
)	O
0	O
.	O
012	O
0	O
.	O
005	O
0	O
.	O
011	O
0	O
.	O
002	O
RMS	O
(	O
angles	O
)	O
(°)	O
1	O
.	O
26	O
0	O
.	O
58	O
1	O
.	O
02	O
0	O
.	O
40	O
Ramachandran	O
favored	O
(%)	O
100	O
99	O
100	O
99	O
Ramachandran	O
allowed	O
(%)	O
0	O
1	O
0	O
1	O
Ramachandran	O
outliers	O
(%)	O
0	O
0	O
0	O
0	O
Clash	O
score	O
1	O
.	O
42	O
1	O
.	O
42	O
1	O
.	O
79	O
0	O
.	O
97	O
Average	O
B	O
-	O
factor	O
(	O
Å2	O
)	O
33	O
.	O
90	O
42	O
.	O
31	O
41	O
.	O
34	O
47	O
.	O
68	O
macromolecules	O
33	O
.	O
80	O
42	O
.	O
35	O
41	O
.	O
31	O
47	O
.	O
60	O
ligands	O
40	O
.	O
40	O
72	O
.	O
80	O
65	O
.	O
55	O
72	O
.	O
34	O
solvent	O
36	O
.	O
20	O
38	O
.	O
95	O
41	O
.	O
46	O
33	O
.	O
85	O
PDB	O
ID	O
5DA5	O
5L89	O
5L8B	O
5L8G	O

Iron	B-chemical
loading	O
capacity	O
of	O
EncFtn	B-protein
,	O
encapsulin	B-protein
and	O
ferritin	B-protein_type
.	O

Protein	O
samples	O
(	O
at	O
8	O
.	O
5	O
µM	O
)	O
including	O
decameric	B-oligomeric_state
EncFtnsH	B-protein
,	O
encapsulin	B-protein
,	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
and	O
apoferritin	B-protein_state
were	O
mixed	O
with	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
(	O
in	O
0	O
.	O
1	O
%	O
(	O
v	O
/	O
v	O
)	O
HCl	B-chemical
)	O
of	O
different	O
concentrations	O
in	O
50	O
mM	O
Tris	O
-	O
HCl	O
(	O
pH	O
8	O
.	O
0	O
),	O
150	O
mM	O
NaCl	B-chemical
buffer	O
at	O
room	O
temperature	O
for	O
3	O
hrs	O
in	O
the	O
air	O
.	O

Protein	O
-	O
Fe	B-chemical
mixtures	O
were	O
centrifuged	O
at	O
13	O
,	O
000	O
x	O
g	O
to	O
remove	O
precipitated	O
material	O
and	O
desalted	O
prior	O
to	O
the	O
Fe	B-chemical
and	O
protein	O
content	O
analysis	O
by	O
ferrozine	B-experimental_method
assay	I-experimental_method
and	O
BCA	B-experimental_method
microplate	I-experimental_method
assay	I-experimental_method
,	O
respectively	O
.	O

Fe	B-chemical
to	O
protein	O
ratio	O
was	O
calculated	O
to	O
indicate	O
the	O
Fe	B-chemical
binding	O
capacity	O
of	O
the	O
protein	O
.	O

Protein	O
stability	O
was	O
compromised	O
at	O
high	O
iron	B-chemical
concentrations	O
;	O
therefore	O
,	O
the	O
highest	O
iron	B-chemical
loading	O
with	O
the	O
least	O
protein	O
precipitation	O
was	O
used	O
to	O
derive	O
the	O
maximum	O
iron	B-chemical
loading	O
capacity	O
per	O
biological	O
assembly	O
(	O
underlined	O
and	O
highlighted	O
in	O
bold	O
).	O

The	O
biological	O
unit	O
assemblies	O
are	O
a	O
decamer	B-oligomeric_state
for	O
EncFtnsH	B-protein
,	O
a	O
60mer	B-oligomeric_state
for	O
encapsulin	B-protein
,	O
a	O
60mer	B-oligomeric_state
of	O
encapsulin	B-protein
loaded	B-protein_state
with	I-protein_state
12	O
copies	O
of	O
decameric	B-oligomeric_state
EncFtn	B-protein
in	O
the	O
complex	O
,	O
and	O
24mer	B-oligomeric_state
for	O
horse	B-taxonomy_domain
spleen	O
apoferritin	B-protein_state
.	O

Errors	O
are	O
quoted	O
as	O
the	O
standard	O
deviation	O
of	O
three	O
technical	O
repeats	O
in	O
both	O
the	O
ferrozine	B-experimental_method
and	I-experimental_method
BCA	I-experimental_method
microplate	I-experimental_method
assays	I-experimental_method
.	O

The	O
proteins	O
used	O
in	O
Fe	B-chemical
loading	O
experiment	O
came	O
from	O
a	O
single	O
preparation	O
.	O

Protein	O
sample	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
loading	O
(	O
µM	O
)	O
Fe	B-chemical
detected	O
by	O
ferrozine	B-experimental_method
assay	I-experimental_method
(	O
µM	O
)	O
Protein	O
detected	O
by	O
BCA	B-experimental_method
microplate	I-experimental_method
assay	I-experimental_method
(	O
µM	O
)	O
Fe	B-chemical
/	O
monomeric	O
protein	O
Maximum	O
Fe	B-chemical
loading	O
per	O
biological	O
assembly	O
unit	O
8	O
.	O
46	O
µM	O
EncFtnsH	B-protein
-	O
10mer	B-oligomeric_state
0	O
4	O
.	O
73	O
±	O
2	O
.	O
32	O
5	O
.	O
26	O
±	O
0	O
.	O
64	O
0	O
.	O
90	O
±	O
0	O
.	O
44	O
39	O
.	O
9	O
9	O
.	O
93	O
±	O
1	O
.	O
20	O
5	O
.	O
36	O
±	O
0	O
.	O
69	O
1	O
.	O
85	O
±	O
0	O
.	O
22	O
84	O
17	O
.	O
99	O
±	O
2	O
.	O
01	O
4	O
.	O
96	O
±	O
0	O
.	O
04	O
3	O
.	O
63	O
±	O
0	O
.	O
41	O
147	O
21	O
.	O
09	O
±	O
1	O
.	O
94	O
4	O
.	O
44	O
±	O
0	O
.	O
21	O
4	O
.	O
75	O
±	O
0	O
.	O
44	O
48	O
±	O
4	O
224	O
28	O
.	O
68	O
±	O
0	O
.	O
30	O
3	O
.	O
73	O
±	O
0	O
.	O
53	O
7	O
.	O
68	O
±	O
0	O
.	O
08	O
301	O
11	O
.	O
27	O
±	O
1	O
.	O
10	O
2	O
.	O
50	O
±	O
0	O
.	O
05	O
4	O
.	O
51	O
±	O
0	O
.	O
44	O
8	O
.	O
50	O
µM	O
Encapsulin	B-protein
0	O
-	O
1	O
.	O
02	O
±	O
0	O
.	O
54	O
8	O
.	O
63	O
±	O
0	O
.	O
17	O
-	O
0	O
.	O
12	O
±	O
0	O
.	O
06	O
224	O
62	O
.	O
24	O
±	O
2	O
.	O
49	O
10	O
.	O
01	O
±	O
0	O
.	O
58	O
6	O
.	O
22	O
±	O
0	O
.	O
35	O
301	O
67	O
.	O
94	O
±	O
3	O
.	O
15	O
8	O
.	O
69	O
±	O
0	O
.	O
42	O
7	O
.	O
81	O
±	O
0	O
.	O
36	O
450	O
107	O
.	O
96	O
±	O
8	O
.	O
88	O
8	O
.	O
50	O
±	O
0	O
.	O
69	O
12	O
.	O
71	O
±	O
1	O
.	O
05	O
700	O
97	O
.	O
51	O
±	O
3	O
.	O
19	O
7	O
.	O
26	O
±	O
0	O
.	O
20	O
13	O
.	O
44	O
±	O
0	O
.	O
44	O
1000	O
308	O
.	O
63	O
±	O
2	O
.	O
06	O
8	O
.	O
42	O
±	O
0	O
.	O
34	O
36	O
.	O
66	O
±	O
0	O
.	O
24	O
2199	O
±	O
15	O
1500	O
57	O
.	O
09	O
±	O
0	O
.	O
90	O
1	O
.	O
44	O
±	O
0	O
.	O
21	O
39	O
.	O
77	O
±	O
0	O
.	O
62	O
2000	O
9	O
.	O
2	O
±	O
1	O
.	O
16	O
0	O
.	O
21	O
±	O
0	O
.	O
14	O
44	O
.	O
73	O
±	O
5	O
.	O
63	O
8	O
.	O
70	O
µM	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
0	O
3	O
.	O
31	O
±	O
1	O
.	O
57	O
6	O
.	O
85	O
±	O
0	O
.	O
07	O
0	O
.	O
48	O
±	O
0	O
.	O
23	O
224	O
116	O
.	O
27	O
±	O
3	O
.	O
74	O
7	O
.	O
63	O
±	O
0	O
.	O
12	O
15	O
.	O
25	O
±	O
0	O
.	O
49	O
301	O
132	O
.	O
86	O
±	O
4	O
.	O
03	O
6	O
.	O
66	O
±	O
0	O
.	O
31	O
19	O
.	O
96	O
±	O
0	O
.	O
61	O
450	O
220	O
.	O
57	O
±	O
27	O
.	O
33	O
6	O
.	O
12	O
±	O
1	O
.	O
07	O
36	O
.	O
06	O
±	O
4	O
.	O
47	O
700	O
344	O
.	O
03	O
±	O
40	O
.	O
38	O
6	O
.	O
94	O
±	O
0	O
.	O
17	O
49	O
.	O
58	O
±	O
5	O
.	O
82	O
1000	O
496	O
.	O
00	O
±	O
38	O
.	O
48	O
7	O
.	O
19	O
±	O
0	O
.	O
08	O
68	O
.	O
94	O
±	O
5	O
.	O
35	O
4137	O
±	O
321	O
1500	O
569	O
.	O
98	O
±	O
73	O
.	O
63	O
5	O
.	O
73	O
±	O
0	O
.	O
03	O
99	O
.	O
44	O
±	O
12	O
.	O
84	O
2000	O
584	O
.	O
30	O
±	O
28	O
.	O
33	O
4	O
.	O
88	O
±	O
0	O
.	O
22	O
119	O
.	O
62	O
±	O
5	O
.	O
80	O
8	O
.	O
50	O
µM	O
Apoferritin	B-protein_state
0	O
3	O
.	O
95	O
±	O
2	O
.	O
26	O
9	O
.	O
37	O
±	O
0	O
.	O
24	O
0	O
.	O
42	O
±	O
0	O
.	O
25	O
42	O
.	O
5	O
10	O
.	O
27	O
±	O
1	O
.	O
12	O
8	O
.	O
27	O
±	O
0	O
.	O
30	O
1	O
.	O
24	O
±	O
0	O
.	O
18	O
212	O
.	O
5	O
44	O
.	O
48	O
±	O
2	O
.	O
76	O
7	O
.	O
85	O
±	O
0	O
.	O
77	O
5	O
.	O
67	O
±	O
0	O
.	O
83	O
637	O
.	O
5	O
160	O
.	O
93	O
±	O
4	O
.	O
27	O
6	O
.	O
76	O
±	O
0	O
.	O
81	O
23	O
.	O
79	O
±	O
3	O
.	O
12	O
571	O
±	O
75	O
1275	O
114	O
.	O
92	O
±	O
3	O
.	O
17	O
3	O
.	O
84	O
±	O
0	O
.	O
30	O
29	O
.	O
91	O
±	O
2	O
.	O
95	O
1700	O
91	O
.	O
40	O
±	O
3	O
.	O
37	O
3	O
.	O
14	O
±	O
0	O
.	O
35	O
29	O
.	O
13	O
±	O
3	O
.	O
86	O

To	O
understand	O
the	O
impact	O
of	O
the	O
mutants	B-protein_state
on	O
the	O
organization	O
and	O
metal	O
binding	O
of	O
the	O
FOC	B-site
,	O
we	O
determined	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structures	I-evidence
of	O
each	O
of	O
the	O
EncFtnsH	B-protein
mutants	B-protein_state
(	O
See	O
Table	O
4	O
for	O
data	O
collection	O
and	O
refinement	O
statistics	O
).	O

The	O
crystal	O
packing	O
of	O
all	O
of	O
the	O
mutants	B-protein_state
in	O
this	O
study	O
is	O
essentially	O
isomorphous	O
to	O
the	O
EncFtnsH	B-protein
structure	B-evidence
.	O

All	O
of	O
the	O
mutants	B-protein_state
display	O
the	O
same	O
decameric	B-oligomeric_state
arrangement	O
in	O
the	O
crystals	B-evidence
as	O
the	O
EncFtnsH	B-protein
structure	B-evidence
,	O
and	O
the	O
monomers	B-oligomeric_state
superimpose	B-experimental_method
with	O
an	O
average	O
RMSDCα	B-evidence
of	O
less	O
than	O
0	O
.	O
2	O
Å	O
.	O

FOC	B-site
dimer	B-site
interface	I-site
of	O
EncFtnsH	B-mutant
-	I-mutant
E32A	I-mutant
mutant	B-protein_state
.	O

(	O
A	O
)	O
Wall	O
-	O
eyed	O
stereo	O
view	O
of	O
the	O
metal	B-site
-	I-site
binding	I-site
dimerization	I-site
interface	I-site
of	O
EncFtnsH	B-mutant
-	I-mutant
E32A	I-mutant
.	O

Protein	O
residues	O
are	O
shown	O
as	O
sticks	O
with	O
blue	O
and	O
green	O
carbons	O
for	O
the	O
different	O
subunits	B-structure_element
.	O

The	O
2mFo	B-evidence
-	I-evidence
DFc	I-evidence
electron	I-evidence
density	I-evidence
map	I-evidence
is	O
shown	O
as	O
a	O
blue	O
mesh	O
contoured	O
at	O
1	O
.	O
5	O
σ	O
.	O

(	O
B	O
)	O
Views	O
of	O
the	O
FOC	B-site
of	O
the	O
EncFtnsH	B-mutant
-	I-mutant
E32Amutant	I-mutant
.	O

FOC	B-site
dimer	I-site
interface	I-site
of	O
EncFtnsH	B-mutant
-	I-mutant
E62A	I-mutant
mutant	B-protein_state
.	O

(	O
A	O
)	O
Wall	O
-	O
eyed	O
stereo	O
view	O
of	O
the	O
metal	B-site
-	I-site
binding	I-site
dimerization	I-site
interface	I-site
of	O
EncFtnsH	B-mutant
-	I-mutant
E62A	I-mutant
.	O

The	O
single	O
coordinated	O
calcium	B-chemical
ion	O
is	O
shown	O
as	O
a	O
grey	O
sphere	O
.	O
(	O
B	O
)	O
Views	O
of	O
the	O
FOC	B-site
of	O
the	O
EncFtnsH	B-mutant
-	I-mutant
E62A	I-mutant
mutant	B-protein_state
.	O

FOC	B-site
dimer	I-site
interface	I-site
of	O
EncFtnsH	B-mutant
-	I-mutant
H65A	I-mutant
mutant	B-protein_state
.	O

(	O
A	O
)	O
Wall	O
-	O
eyed	O
stereo	O
view	O
of	O
the	O
metal	B-site
-	I-site
binding	I-site
dimerization	I-site
interface	I-site
of	O
EncFtnsH	B-mutant
-	I-mutant
H65A	I-mutant
.	O

The	O
coordinated	O
calcium	B-chemical
ions	O
are	O
shown	O
as	O
a	O
grey	O
spheres	O
with	O
coordination	B-bond_interaction
distances	O
in	O
the	O
FOC	B-site
highlighted	O
with	O
yellow	O
dashed	O
lines	O
.	O

(	O
B	O
)	O
Views	O
of	O
the	O
FOC	B-site
of	O
the	O
EncFtnsH	B-mutant
-	I-mutant
H65A	I-mutant
mutant	B-protein_state
.	O

Comparison	O
of	O
the	O
EncFtnsH	B-protein
FOC	B-site
mutants	B-protein_state
vs	O
wild	B-protein_state
type	I-protein_state
.	O

The	O
structures	B-evidence
of	O
the	O
three	O
EncFtnsH	B-protein
mutants	B-protein_state
were	O
all	O
determined	O
by	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
.	O

The	O
E32A	B-mutant
,	O
E62A	B-mutant
and	O
H65A	B-mutant
mutants	B-protein_state
were	O
crystallized	B-experimental_method
in	O
identical	O
conditions	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
.	O

EncFtnsH	B-protein
structure	B-evidence
and	O
were	O
essentially	O
isomorphous	O
in	O
terms	O
of	O
their	O
unit	O
cell	O
dimensions	O
.	O

The	O
FOC	B-site
residues	O
of	O
the	O
mutants	B-protein_state
and	O
native	B-protein_state
EncFtnsH	B-protein
structures	B-evidence
are	O
shown	O
as	O
sticks	O
with	O
coordinated	B-bond_interaction
Fe2	B-chemical
+	I-chemical
as	O
orange	O
and	O
Ca2	B-chemical
+	I-chemical
as	O
grey	O
spheres	O
and	O
are	O
colored	O
as	O
follows	O
:	O
wild	B-protein_state
type	I-protein_state
,	O
grey	O
;	O
E32A	B-mutant
,	O
pink	O
;	O
E62A	B-mutant
,	O
green	O
;	O
H65A	B-mutant
,	O
blue	O
.	O

Of	O
the	O
mutants	B-protein_state
,	O
only	O
H65A	B-mutant
has	O
any	O
coordinated	B-bond_interaction
metal	O
ions	O
,	O
which	O
appear	O
to	O
be	O
calcium	B-chemical
ions	O
from	O
the	O
crystallization	O
condition	O
.	O

The	O
overall	O
organization	O
of	O
FOC	B-site
residues	O
is	O
retained	O
in	O
the	O
mutants	B-protein_state
,	O
with	O
almost	O
no	O
backbone	O
movements	O
.	O

Significant	O
differences	O
center	O
around	O
Tyr39	B-residue_name_number
,	O
which	O
moves	O
to	O
coordinate	B-bond_interaction
the	O
bound	B-protein_state
calcium	B-chemical
ions	O
in	O
the	O
H65A	B-mutant
mutant	B-protein_state
;	O
and	O
Glu32	B-residue_name_number
,	O
which	O
moves	O
away	O
from	O
the	O
metal	O
ions	O
in	O
this	O
structure	B-evidence
.	O

Close	O
inspection	O
of	O
the	O
region	O
of	O
the	O
protein	O
around	O
the	O
FOC	B-site
in	O
each	O
of	O
the	O
mutants	B-protein_state
highlights	O
their	O
effect	O
on	O
metal	O
binding	O
(	O
Figure	O
11	O
and	O
Figure	O
11	O
—	O
figure	O
supplement	O
1	O
–	O
3	O
).	O

In	O
the	O
E32A	B-mutant
mutant	B-protein_state
the	O
position	O
of	O
the	O
side	O
chains	O
of	O
the	O
remaining	O
iron	B-site
coordinating	I-site
residues	I-site
in	O
the	O
FOC	B-site
is	O
essentially	O
unchanged	O
,	O
but	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
axial	O
-	O
metal	O
coordinating	B-bond_interaction
ligand	O
provided	O
by	O
the	O
Glu32	B-residue_name_number
side	O
chain	O
abrogates	B-protein_state
metal	I-protein_state
binding	I-protein_state
in	O
this	O
site	O
.	O

The	O
Glu31	B-site
/	I-site
34	I-site
-	I-site
site	I-site
also	O
lacks	B-protein_state
metal	B-chemical
,	O
with	O
the	O
side	O
chain	O
of	O
Glu31	B-residue_name_number
rotated	O
by	O
180	O
°	O
at	O
the	O
Cβ	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
metal	B-chemical
(	O
Figure	O
11	O
—	O
figure	O
supplement	O
1	O
).	O

The	O
E62A	B-mutant
mutant	B-protein_state
has	O
a	O
similar	O
effect	O
on	O
the	O
FOC	B-site
to	O
the	O
E32A	B-mutant
mutant	B-protein_state
,	O
however	O
the	O
entry	B-site
site	I-site
still	O
has	O
a	O
calcium	B-chemical
ion	O
coordinated	B-bond_interaction
between	O
residues	O
Glu31	B-residue_name_number
and	O
Glu34	B-residue_name_number
(	O
Figure	O
11	O
—	O
figure	O
supplement	O
2	O
).	O

The	O
H65A	B-mutant
mutant	B-protein_state
diverges	O
significantly	O
from	O
the	O
wild	B-protein_state
type	I-protein_state
in	O
the	O
position	O
of	O
the	O
residues	O
Glu32	B-residue_name_number
and	O
Tyr39	B-residue_name_number
in	O
the	O
FOC	B-site
.	O

E32	B-residue_name_number
appears	O
in	O
either	O
the	O
original	O
orientation	O
as	O
the	O
wild	B-protein_state
type	I-protein_state
and	O
coordinates	B-bond_interaction
Ca2	B-chemical
+	I-chemical
in	O
this	O
position	O
,	O
or	O
it	O
is	O
flipped	O
by	O
180	O
°	O
at	O
the	O
Cβ	O
,	O
moving	O
away	O
from	O
the	O
coordinated	B-bond_interaction
calcium	B-chemical
ion	O
in	O
the	O
FOC	B-site
.	O

Tyr39	B-residue_name_number
moves	O
closer	O
to	O
Ca2	B-chemical
+	I-chemical
compared	O
to	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
coordinates	B-bond_interaction
the	O
calcium	B-chemical
ion	O
(	O
Figure	O
11	O
—	O
figure	O
supplement	O
3	O
).	O

A	O
single	O
calcium	B-chemical
ion	O
is	O
present	O
in	O
the	O
entry	B-site
site	I-site
of	O
this	O
mutant	B-protein_state
;	O
however	O
,	O
Glu31	B-residue_name_number
of	O
one	O
chain	O
is	O
rotated	O
away	O
from	O
the	O
metal	O
ion	O
and	O
is	O
not	O
involved	O
in	O
coordination	B-bond_interaction
.	O

Taken	O
together	O
the	O
results	O
of	O
our	O
data	O
show	O
that	O
these	O
changes	O
to	O
the	O
FOC	B-site
of	O
EncFtn	B-protein
still	O
permit	O
the	O
formation	O
of	O
the	O
decameric	B-oligomeric_state
form	O
of	O
the	O
protein	O
.	O

While	O
the	O
proteins	O
all	O
appear	O
decameric	B-oligomeric_state
in	O
crystals	B-evidence
,	O
their	O
solution	O
and	O
gas	O
-	O
phase	O
behavior	O
differs	O
considerably	O
and	O
the	O
mutants	B-protein_state
no	O
longer	O
show	O
metal	O
-	O
dependent	O
oligomerization	O
.	O

These	O
results	O
highlight	O
the	O
importance	O
of	O
metal	B-chemical
coordination	B-bond_interaction
in	O
the	O
FOC	B-site
for	O
the	O
stability	O
and	O
assembly	O
of	O
the	O
EncFtn	B-protein
protein	O
.	O

Progress	B-evidence
curves	I-evidence
recording	O
ferroxidase	B-protein_type
activity	O
of	O
EncFtnsH	B-protein
mutants	B-protein_state
.	O

20	O
µM	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
EncFtnsH	B-protein
,	O
E32A	B-mutant
,	O
E62A	B-mutant
and	O
H65A	B-mutant
mutants	B-protein_state
were	O
mixed	O
with	O
20	O
µM	O
or	O
100	O
µM	O
acidic	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
,	O
respectively	O
.	O

Absorbance	O
at	O
315	O
nm	O
was	O
recorded	O
for	O
1800	O
s	O
at	O
25	O
°	O
C	O
as	O
an	O
indication	O
of	O
Fe3	B-chemical
+	I-chemical
formation	O
.	O

Protein	O
free	O
samples	O
(	O
dashed	O
and	O
dotted	O
lines	O
)	O
were	O
measured	O
for	O
Fe2	B-chemical
+	I-chemical
background	O
oxidation	O
as	O
controls	O
.	O

Relative	O
ferroxidase	B-protein_type
activity	O
of	O
EncFtnsH	B-protein
mutants	B-protein_state
.	O

EncFtnsH	B-protein
,	O
and	O
the	O
mutant	B-protein_state
forms	O
E32A	B-mutant
,	O
E62A	B-mutant
and	O
H65A	B-mutant
,	O
each	O
at	O
20	O
µM	O
,	O
were	O
mixed	O
with	O
100	O
µM	O
acidic	O
Fe	B-chemical
(	I-chemical
NH4	I-chemical
)	I-chemical
2	I-chemical
(	I-chemical
SO4	I-chemical
)	I-chemical
2	I-chemical
.	O

Ferroxidase	B-protein_type
activity	O
of	O
the	O
mutant	B-protein_state
forms	O
is	O
determined	O
by	O
measuring	B-experimental_method
the	I-experimental_method
absorbance	I-experimental_method
at	I-experimental_method
315	I-experimental_method
nm	I-experimental_method
for	O
1800	O
s	O
at	O
25	O
°	O
C	O
as	O
an	O
indication	O
of	O
Fe3	B-chemical
+	I-chemical
formation	O
.	O

The	O
relative	O
ferroxidase	B-protein_type
activity	O
of	O
mutants	B-protein_state
is	O
plotted	O
as	O
a	O
proportion	O
of	O
the	O
activity	O
of	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
protein	O
using	O
the	O
endpoint	O
measurement	B-experimental_method
of	I-experimental_method
A315	I-experimental_method
.	O

The	O
FOC	B-site
mutants	B-protein_state
showed	O
reduced	O
ferroxidase	B-protein_type
activity	O
to	O
varied	O
extents	O
,	O
among	O
which	O
E62A	B-mutant
significantly	O
abrogated	O
the	O
ferroxidase	B-protein_type
activity	O
.	O

To	O
address	O
the	O
question	O
of	O
how	O
mutagenesis	B-experimental_method
of	O
the	O
iron	B-site
coordinating	I-site
residues	I-site
affects	O
the	O
enzymatic	O
activity	O
of	O
the	O
EncFtnsH	B-protein
protein	O
we	O
recorded	O
progress	B-evidence
curves	I-evidence
for	O
the	O
oxidation	O
of	O
Fe2	B-chemical
+	I-chemical
to	O
Fe3	B-chemical
+	I-chemical
by	O
the	O
different	O
mutants	B-protein_state
as	O
before	O
.	O

Mutagenesis	B-experimental_method
of	O
E32A	B-mutant
and	O
H65A	B-mutant
reduces	O
the	O
activity	O
of	O
EncFtnsH	B-protein
by	O
about	O
40	O
%-	O
55	O
%;	O
the	O
E62A	B-mutant
mutant	B-protein_state
completely	O
abrogates	O
activity	O
,	O
presumably	O
through	O
the	O
loss	B-protein_state
of	I-protein_state
the	O
bridging	O
coordination	B-bond_interaction
for	O
the	O
formation	O
of	O
the	O
di	B-site
-	I-site
nuclear	I-site
iron	I-site
center	I-site
of	O
the	O
FOC	B-site
(	O
Figure	O
12	O
).	O

Collectively	O
,	O
the	O
effect	O
of	O
mutating	B-experimental_method
these	O
residues	O
in	O
the	O
FOC	B-site
confirms	O
the	O
importance	O
of	O
the	O
iron	B-site
coordinating	I-site
residues	I-site
for	O
the	O
ferroxidase	B-protein_type
activity	O
of	O
the	O
EncFtnsH	B-protein
protein	O
.	O

Phylogenetic	B-evidence
tree	I-evidence
of	O
ferritin	B-protein_type
family	O
proteins	O
.	O

The	O
tree	O
was	O
built	O
using	O
the	O
Neighbor	B-experimental_method
-	I-experimental_method
Joining	I-experimental_method
method	I-experimental_method
based	O
on	O
step	B-experimental_method
-	I-experimental_method
wise	I-experimental_method
amino	I-experimental_method
acid	I-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
the	O
four	B-structure_element
-	I-structure_element
helical	I-structure_element
bundle	I-structure_element
portions	O
of	O
ferritin	B-protein_type
family	O
proteins	O
(	O
Supplementary	O
file	O
1	O
).	O

The	O
evolutionary	B-evidence
distances	I-evidence
were	O
computed	O
using	O
the	O
p	B-experimental_method
-	I-experimental_method
distance	I-experimental_method
method	I-experimental_method
and	O
are	O
in	O
the	O
units	O
of	O
the	O
number	O
of	O
amino	O
acid	O
differences	O
per	O
site	O
.	O

Our	O
study	O
reports	O
on	O
a	O
new	O
class	O
of	O
ferritin	B-protein_type
-	O
like	O
proteins	O
(	O
EncFtn	B-protein
),	O
which	O
are	O
associated	O
with	O
bacterial	B-taxonomy_domain
encapsulin	B-protein
nanocompartments	B-complex_assembly
(	O
Enc	B-protein
).	O

By	O
studying	O
the	O
EncFtn	B-protein
from	O
R	B-species
.	I-species
rubrum	I-species
we	O
demonstrate	O
that	O
iron	B-chemical
binding	O
results	O
in	O
assembly	O
of	O
EncFtn	B-protein
decamers	B-oligomeric_state
,	O
which	O
display	O
a	O
unique	O
annular	O
architecture	O
.	O

Despite	O
a	O
radically	O
different	O
quaternary	O
structure	O
to	O
the	O
classical	B-protein_state
ferritins	B-protein_type
,	O
the	O
four	B-structure_element
-	I-structure_element
helical	I-structure_element
bundle	I-structure_element
scaffold	I-structure_element
and	O
FOC	B-site
of	O
EncFtnsH	B-protein
are	O
strikingly	O
similar	O
to	O
ferritin	B-protein_type
(	O
Figure	O
6A	O
).	O

A	O
sequence	B-experimental_method
-	I-experimental_method
based	I-experimental_method
phylogenetic	I-experimental_method
tree	I-experimental_method
for	O
proteins	O
in	O
the	O
ferritin	B-protein_type
family	O
was	O
constructed	O
;	O
in	O
addition	O
to	O
the	O
classical	B-protein_state
ferritins	B-protein_type
,	O
bacterioferritins	B-protein_type
and	O
Dps	B-protein_type
proteins	O
,	O
our	O
analysis	O
included	O
the	O
encapsulin	B-protein_type
-	I-protein_type
associated	I-protein_type
ferritin	I-protein_type
-	I-protein_type
like	I-protein_type
proteins	I-protein_type
(	O
EncFtns	B-protein_type
)	O
and	O
a	O
group	O
related	O
to	O
these	O
,	O
but	O
lacking	O
the	O
encapsulin	B-protein
sequence	O
(	O
Non	B-protein_type
-	I-protein_type
EncFtn	I-protein_type
).	O

The	O
analysis	O
revealed	O
that	O
the	O
EncFtn	B-protein
and	O
Non	B-protein_type
-	I-protein_type
EncFtn	I-protein_type
proteins	O
form	O
groups	O
distinct	O
from	O
the	O
other	O
clearly	O
delineated	O
groups	O
of	O
ferritins	B-protein_type
,	O
and	O
represent	O
outliers	O
in	O
the	O
tree	O
(	O
Figure	O
13	O
).	O

While	O
it	O
is	O
difficult	O
to	O
infer	O
ancestral	O
lineages	O
in	O
protein	O
families	O
,	O
the	O
similarity	O
seen	O
in	O
the	O
active	B-site
site	I-site
scaffold	I-site
of	O
these	O
proteins	O
highlights	O
a	O
shared	O
evolutionary	O
relationship	O
between	O
EncFtn	B-protein_type
proteins	O
and	O
other	O
members	O
of	O
the	O
ferritin	B-protein_type
superfamily	O
that	O
has	O
been	O
noted	O
in	O
previous	O
studies	O
(;	O
).	O

From	O
this	O
analysis	O
,	O
we	O
propose	O
that	O
the	O
four	B-structure_element
-	I-structure_element
helical	I-structure_element
fold	I-structure_element
of	O
the	O
classical	B-protein_state
ferritins	B-protein_type
may	O
have	O
arisen	O
through	O
gene	O
duplication	O
of	O
an	O
ancestor	O
of	O
EncFtn	B-protein
.	O

This	O
gene	O
duplication	O
would	O
result	O
in	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
one	O
EncFtn	B-protein
monomer	B-oligomeric_state
being	O
linked	O
to	O
the	O
N	O
-	O
terminus	O
of	O
another	O
and	O
thus	O
stabilizing	O
the	O
four	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
fold	I-structure_element
within	O
a	O
single	O
polypeptide	O
chain	O
(	O
Figure	O
6B	O
).	O

Linking	O
the	O
protein	O
together	O
in	O
this	O
way	O
relaxes	O
the	O
requirement	O
for	O
the	O
maintenance	O
of	O
a	O
symmetrical	O
FOC	B-site
and	O
thus	O
provides	O
a	O
path	O
to	O
the	O
diversity	O
in	O
active	B-site
-	I-site
site	I-site
residues	I-site
seen	O
across	O
the	O
ferritin	B-protein_type
family	O
(	O
Figure	O
6A	O
,	O
residues	O
Glu95	B-residue_name_number
,	O
Gln128	B-residue_name_number
and	O
Glu131	B-residue_name_number
in	O
PmFtn	B-protein
,	O
Supplementary	O
file	O
1	O
).	O

Relationship	O
between	O
ferritin	B-protein_type
structure	B-evidence
and	O
activity	O

The	O
quaternary	O
arrangement	O
of	O
classical	B-protein_state
ferritins	B-protein_type
into	O
an	O
octahedral	B-protein_state
nanocage	B-complex_assembly
and	O
Dps	B-protein
into	O
a	O
dodecamer	B-oligomeric_state
is	O
absolutely	O
required	O
for	O
their	O
function	O
as	O
iron	B-chemical
storage	O
compartments	O
.	O

The	O
oxidation	O
and	O
mineralization	O
of	O
iron	B-chemical
must	O
be	O
spatially	O
separated	O
from	O
the	O
host	O
cytosol	O
to	O
prevent	O
the	O
formation	O
of	O
damaging	O
hydroxyl	O
radicals	O
in	O
the	O
Fenton	O
and	O
Haber	O
-	O
Weiss	O
reactions	O
.	O

This	O
is	O
achieved	O
in	O
all	O
ferritins	B-protein_type
by	O
confining	O
the	O
oxidation	O
of	O
iron	B-chemical
to	O
the	O
interior	O
of	O
the	O
protein	O
complex	O
,	O
thus	O
achieving	O
sequestration	O
of	O
the	O
Fe3	B-chemical
+	I-chemical
mineralization	O
product	O
.	O

A	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
FOC	B-site
of	O
EncFtn	B-protein
with	O
the	O
classical	B-protein_state
ferritin	B-protein_type
PmFtn	B-protein
shows	O
that	O
the	O
central	B-structure_element
ring	I-structure_element
of	O
EncFtn	B-protein
corresponds	O
to	O
the	O
external	O
surface	O
of	O
ferritin	B-protein_type
,	O
while	O
the	O
outer	O
circumference	O
of	O
EncFtn	B-protein
is	O
congruent	O
with	O
the	O
inner	O
mineralization	B-site
surface	I-site
of	O
ferritin	B-protein_type
(	O
Figure	O
6	O
—	O
figure	O
supplement	O
1A	O
).	O

This	O
overlay	B-experimental_method
highlights	O
the	O
fact	O
that	O
the	O
ferroxidase	B-site
center	I-site
of	O
EncFtn	B-protein
faces	O
in	O
the	O
opposite	O
direction	O
relative	O
to	O
the	O
classical	B-protein_state
ferritins	B-protein_type
and	O
is	O
essentially	O
inside	O
out	O
regarding	O
iron	B-chemical
storage	O
space	O
(	O
Figure	O
6	O
—	O
figure	O
supplement	O
1B	O
,	O
boxed	O
region	O
).	O

Analysis	O
of	O
each	O
of	O
the	O
single	O
mutations	B-experimental_method
(	O
E32A	B-mutant
,	O
E62A	B-mutant
and	O
H65A	B-mutant
)	O
made	O
in	O
the	O
FOC	B-site
highlights	O
the	O
importance	O
of	O
the	O
iron	B-site
-	I-site
coordinating	I-site
residues	I-site
in	O
the	O
catalytic	O
activity	O
of	O
EncFtn	B-protein
.	O

Furthermore	O
,	O
the	O
position	O
of	O
the	O
calcium	B-chemical
ion	O
coordinated	B-bond_interaction
by	I-bond_interaction
Glu31	B-residue_name_number
and	O
Glu34	B-residue_name_number
seen	O
in	O
the	O
EncFtnsH	B-protein
structure	B-evidence
suggests	O
an	O
entry	B-site
site	I-site
to	O
channel	O
metal	O
ions	O
into	O
the	O
FOC	B-site
;	O
we	O
propose	O
that	O
this	O
site	O
binds	O
hydrated	O
iron	B-chemical
ions	O
in	O
vivo	O
and	O
acts	O
as	O
a	O
selectivity	O
filter	O
and	O
gate	O
for	O
the	O
FOC	B-site
.	O

The	O
constellation	O
of	O
charged	O
residues	O
on	O
the	O
outer	O
circumference	O
of	O
EncFtn	B-protein
(	O
His57	B-residue_name_number
,	O
Glu61	B-residue_name_number
and	O
Glu64	B-residue_name_number
)	O
could	O
function	O
in	O
the	O
same	O
way	O
as	O
the	O
residues	O
lining	O
the	O
mineralization	B-site
surface	I-site
within	O
the	O
classical	B-protein_state
ferritin	B-protein_type
nanocage	B-complex_assembly
,	O
and	O
given	O
their	O
proximity	O
to	O
the	O
FOC	B-site
these	O
sites	O
may	O
be	O
the	O
exit	B-site
portal	I-site
and	O
mineralization	B-site
site	I-site
.	O

The	O
absolute	O
requirement	O
for	O
the	O
spatial	O
separation	O
of	O
oxidation	O
and	O
mineralization	O
in	O
ferritins	B-protein_type
suggests	O
that	O
the	O
EncFtn	B-protein_type
family	O
proteins	O
are	O
not	O
capable	O
of	O
storing	O
iron	B-chemical
minerals	O
due	O
to	O
the	O
absence	B-protein_state
of	I-protein_state
an	O
enclosed	O
compartment	O
in	O
their	O
structure	O
(	O
Figure	O
6	O
—	O
figure	O
supplement	O
1B	O
).	O

Our	O
biochemical	B-experimental_method
characterization	I-experimental_method
of	O
EncFtn	B-protein
supports	O
this	O
hypothesis	O
,	O
indicating	O
that	O
while	O
this	O
protein	O
is	O
capable	O
of	O
oxidizing	O
iron	B-chemical
,	O
it	O
does	O
not	O
accrue	O
mineralized	O
iron	B-chemical
in	O
an	O
analogous	O
manner	O
to	O
classical	B-protein_state
ferritins	B-protein_type
.	O

While	O
EncFtn	B-protein
does	O
not	O
store	O
iron	B-chemical
itself	O
,	O
its	O
association	O
with	O
the	O
encapsulin	B-protein
nanocage	B-complex_assembly
suggests	O
that	O
mineralization	O
occurs	O
within	O
the	O
cavity	B-site
of	O
the	O
encapsulin	B-protein
shell	B-structure_element
.	O

Our	O
ferroxidase	B-experimental_method
assay	I-experimental_method
data	O
on	O
the	O
recombinant	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartments	B-complex_assembly
,	O
which	O
accrue	O
over	O
4100	O
iron	B-chemical
ions	O
per	O
complex	O
and	O
form	O
regular	O
nanoparticles	B-complex_assembly
,	O
are	O
consistent	O
with	O
the	O
encapsulin	B-protein
protein	O
acting	O
as	O
the	O
store	O
for	O
iron	B-chemical
oxidized	O
by	O
the	O
EncFtn	B-protein
enzyme	O
.	O

TEM	B-experimental_method
analysis	O
of	O
the	O
reaction	O
products	O
shows	O
the	O
production	O
of	O
homogeneous	O
iron	B-chemical
nanoparticles	O
only	O
in	O
the	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartment	B-complex_assembly
(	O
Figure	O
8	O
—	O
figure	O
supplement	O
1	O
).	O

Model	O
of	O
iron	B-chemical
oxidation	O
in	O
encapsulin	B-protein
nanocompartments	B-complex_assembly
.	O

(	O
A	O
)	O
Model	O
of	O
EncFtnsH	B-protein
docking	B-experimental_method
to	O
the	O
encapsulin	B-protein
shell	B-structure_element
.	O

A	O
single	O
pentamer	B-oligomeric_state
of	O
the	O
icosahedral	B-protein_state
T	B-species
.	I-species
maritima	I-species
encapsulin	B-protein
structure	B-evidence
(	O
PDBID	O
:	O
3DKT	O
)	O
is	O
shown	O
as	O
a	O
blue	O
surface	O
with	O
the	O
encapsulin	B-protein
localization	B-structure_element
sequence	I-structure_element
of	O
EncFtn	B-protein
shown	O
as	O
a	O
purple	O
surface	O
.	O

The	O
C	O
-	O
terminal	O
regions	O
of	O
the	O
EncFtn	B-protein
subunits	B-structure_element
correspond	O
to	O
the	O
position	O
of	O
the	O
localization	B-structure_element
sequences	I-structure_element
seen	O
in	O
3DKT	O
.	O

Alignment	B-experimental_method
of	O
EncFtnsH	B-protein
with	O
3DKT	O
positions	O
the	O
central	B-site
channel	I-site
directly	O
above	O
the	O
pore	B-site
in	O
the	O
3DKT	O
pentamer	B-oligomeric_state
axis	O
(	O
shown	O
as	O
a	O
grey	O
pentagon	O
).	O
(	O
B	O
)	O
Surface	O
view	O
of	O
EncFtn	B-protein
within	O
the	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
(	O
grey	O
and	O
blue	O
respectively	O
).	O

The	O
lumen	O
of	O
the	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
is	O
considerably	O
larger	O
than	O
the	O
interior	O
of	O
ferritin	B-protein_type
(	O
shown	O
in	O
orange	O
behind	O
the	O
encapsulin	B-protein
for	O
reference	O
)	O
and	O
thus	O
allows	O
the	O
storage	O
of	O
significantly	O
more	O
iron	B-chemical
.	O

The	O
proposed	O
pathway	O
for	O
iron	B-chemical
movement	O
through	O
the	O
encapsulin	B-protein
shell	B-structure_element
and	O
EncFtn	B-protein
FOC	B-site
is	O
shown	O
with	O
arrows	O
.	O
(	O
C	O
)	O
Model	O
ofiron	O
oxidation	O
within	O
an	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
.	O

As	O
EncFtn	B-protein
is	O
unable	O
to	O
mineralize	O
iron	B-chemical
on	O
its	O
surface	O
directly	O
,	O
Fe2	B-chemical
+	I-chemical
must	O
pass	O
through	O
the	O
encapsulin	B-protein
shell	B-structure_element
to	O
access	O
the	O
first	O
metal	B-site
binding	I-site
site	I-site
within	O
the	O
central	B-site
channel	I-site
of	O
EncFtnsH	B-protein
(	O
entry	B-site
site	I-site
)	O
prior	O
to	O
oxidation	O
within	O
the	O
FOC	B-site
and	O
release	O
as	O
Fe3	B-chemical
+	I-chemical
to	O
the	O
outer	O
surface	O
of	O
the	O
protein	O
where	O
it	O
can	O
be	O
mineralized	O
within	O
the	O
lumen	O
of	O
the	O
encapsulin	B-protein
cage	O
.	O

Docking	B-experimental_method
the	O
decamer	B-oligomeric_state
structure	B-evidence
of	O
EncFtnsH	B-protein
into	O
the	O
pentamer	B-oligomeric_state
of	O
the	O
T	B-species
.	I-species
maritima	I-species
encapsulin	B-protein
Tmari_0786	B-gene
(	O
PDB	O
ID	O
:	O
3DKT	O
)	O
shows	O
that	O
the	O
position	O
of	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
extensions	I-structure_element
of	O
our	O
EncFtnsH	B-protein
structure	B-evidence
are	O
consistent	O
with	O
the	O
localization	B-structure_element
sequences	I-structure_element
seen	O
bound	B-protein_state
to	I-protein_state
the	O
encapsulin	B-protein
protein	O
(	O
Figure	O
14A	O
).	O

Thus	O
,	O
it	O
appears	O
that	O
the	O
EncFtn	B-protein
decamer	B-oligomeric_state
is	O
the	O
physiological	O
state	O
of	O
this	O
protein	O
.	O

This	O
arrangement	O
positions	O
the	O
central	B-structure_element
ring	I-structure_element
of	O
EncFtn	B-protein
directly	O
above	O
the	O
pore	B-site
at	O
the	O
five	O
-	O
fold	O
symmetry	O
axis	O
of	O
the	O
encapsulin	B-protein
shell	B-structure_element
and	O
highlights	O
a	O
potential	O
route	O
for	O
the	O
entry	O
of	O
iron	B-chemical
into	O
the	O
encapsulin	B-protein
and	O
towards	O
the	O
active	B-site
site	I-site
of	O
EncFtn	B-protein
.	O

A	O
comparison	O
of	O
the	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
and	O
the	O
ferritin	B-protein_type
nanocage	B-complex_assembly
highlights	O
the	O
size	O
differential	O
between	O
the	O
two	O
complexes	O
(	O
Figure	O
14B	O
)	O
that	O
allows	O
the	O
encapsulin	B-protein
to	O
store	O
significantly	O
more	O
iron	B-chemical
.	O

The	O
presence	B-protein_state
of	I-protein_state
five	O
FOCs	B-site
per	O
EncFtnsH	B-protein
decamer	B-oligomeric_state
and	O
the	O
fact	O
that	O
the	O
icosahedral	B-protein_state
encapsulin	B-protein
nanocage	B-complex_assembly
can	O
hold	O
up	O
to	O
twelve	O
of	O
decameric	B-oligomeric_state
EncFtn	B-protein
between	O
each	O
of	O
the	O
internal	O
five	O
-	O
fold	O
vertices	O
means	O
that	O
they	O
can	O
achieve	O
a	O
high	O
rate	O
of	O
iron	B-chemical
mineralization	O
across	O
the	O
entire	O
nanocompartment	B-complex_assembly
.	O

This	O
arrangement	O
of	O
multiple	O
reaction	O
centers	O
in	O
a	O
single	O
protein	O
assembly	O
is	O
reminiscent	O
of	O
classical	B-protein_state
ferritins	B-protein_type
,	O
which	O
has	O
24	O
FOCs	B-site
distributed	O
around	O
the	O
nanocage	B-complex_assembly
.	O

Our	O
structural	B-evidence
data	I-evidence
,	O
coupled	O
with	O
biochemical	B-experimental_method
and	I-experimental_method
ICP	I-experimental_method
-	I-experimental_method
MS	I-experimental_method
analysis	O
,	O
suggest	O
a	O
model	O
for	O
the	O
activity	O
of	O
the	O
encapsulin	B-protein
iron	B-complex_assembly
-	I-complex_assembly
megastore	I-complex_assembly
(	O
Figure	O
14C	O
).	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
T	B-species
.	I-species
maritima	I-species
encapsulin	B-protein
shell	B-structure_element
protein	O
has	O
a	O
negatively	B-site
charged	I-site
pore	I-site
positioned	O
to	O
allow	O
the	O
passage	O
of	O
Fe2	B-chemical
+	I-chemical
into	O
the	O
encapsulin	B-protein
and	O
directs	O
the	O
metal	O
towards	O
the	O
central	O
,	O
negatively	B-site
charged	I-site
hole	I-site
of	O
the	O
EncFtn	B-protein
ring	B-structure_element
(	O
Figure	O
4	O
—	O
figure	O
supplement	O
1	O
).	O

The	O
five	O
metal	B-site
-	I-site
binding	I-site
sites	I-site
on	O
the	O
interior	O
of	O
the	O
ring	B-structure_element
(	O
Glu31	B-site
/	I-site
34	I-site
-	I-site
sites	I-site
)	O
may	O
select	O
for	O
the	O
Fe2	B-chemical
+	I-chemical
ion	O
and	O
direct	O
it	O
towards	O
their	O
cognate	O
FOCs	B-site
.	O

We	O
propose	O
that	O
the	O
oxidation	O
of	O
Fe2	B-chemical
+	I-chemical
to	O
Fe3	B-chemical
+	I-chemical
occurs	O
within	O
the	O
FOC	B-site
according	O
to	O
the	O
model	O
postulated	O
by	O
in	O
which	O
the	O
FOC	B-site
acts	O
as	O
a	O
substrate	B-site
site	I-site
through	O
which	O
iron	B-chemical
passes	O
and	O
is	O
released	O
on	O
to	O
weakly	B-site
coordinating	I-site
sites	I-site
at	O
the	O
outer	O
circumference	O
of	O
the	O
protein	O
(	O
His57	B-residue_name_number
,	O
Glu61	B-residue_name_number
and	O
Glu64	B-residue_name_number
),	O
where	O
it	O
is	O
able	O
to	O
form	O
ferrihydrite	B-chemical
minerals	O
which	O
can	O
be	O
safely	O
deposited	O
within	O
the	O
lumen	O
of	O
the	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
(	O
Figure	O
14	O
).	O

Here	O
we	O
describe	O
for	O
the	O
first	O
time	O
the	O
structure	B-evidence
and	O
biochemistry	O
of	O
a	O
new	O
class	O
of	O
encapsulin	B-protein_type
-	I-protein_type
associated	I-protein_type
ferritin	I-protein_type
-	I-protein_type
like	I-protein_type
protein	I-protein_type
and	O
demonstrate	O
that	O
it	O
has	O
an	O
absolute	O
requirement	O
for	O
compartmentalization	O
within	O
an	O
encapsulin	B-protein
nanocage	B-complex_assembly
to	O
act	O
as	O
an	O
iron	B-chemical
store	O
.	O

Further	O
work	O
on	O
the	O
EncFtn	B-complex_assembly
-	I-complex_assembly
Enc	I-complex_assembly
nanocompartment	B-complex_assembly
will	O
establish	O
the	O
structural	O
basis	O
for	O
the	O
movement	O
of	O
iron	B-chemical
through	O
the	O
encapsulin	B-protein
shell	B-structure_element
,	O
the	O
mechanism	O
of	O
iron	B-chemical
oxidation	O
by	O
the	O
EncFtn	B-protein
FOC	B-site
and	O
its	O
subsequent	O
storage	O
in	O
the	O
lumen	O
of	O
the	O
encapsulin	B-protein
nanocompartment	B-complex_assembly
.	O