diff --git "a/annotation_IOB/PMC5012862.tsv" "b/annotation_IOB/PMC5012862.tsv"
new file mode 100644--- /dev/null
+++ "b/annotation_IOB/PMC5012862.tsv"
@@ -0,0 +1,15440 @@
+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
+