interstrand crosslinks ( icls ) are toxic dna lesions that prevent the separation of the two filaments in the dna double helix , impairing dna replication and transcription .
the fanconi anemia ( fa)/brca pathway is the major interstrand crosslink repair process in higher eukaryotes ( knipscheer et  al . , 2009 , kottemann and smogorzewska , 2013 ) .
activation of this pathway depends on the specific mono - ubiquitination of the fanci and fancd2 proteins by the fa core complex ( ceccaldi et  al . , 2016 , garcia - higuera et  al . , 2001 ,
mutations in the fa genes cause a rare genetic disorder characterized by hypersensitivity to crosslinking agents , chromosomal instability , and cancer predisposition ( deans and west , 2011 ) . despite the enormous effort of several research groups , a mechanistic understanding of the activation of the fa icl repair pathway is only starting to emerge ( walden and deans , 2014 ) . a key player in the recognition of icls is the fa anchor complex ( consisting of the fancm dna translocase , faap24 , and two histone - like factors named mhf1 - 2 ) , which , in turn , recruits the fa core complex to the dna damage site ( ciccia et  al .
, 2007 , coulthard et  al . , 2013 , kim et  al . , 2008 , yan et  al . , 2010 ) .
the  core complex is composed of three distinct protein assemblies with non - redundant functions : fancb - fancl - faap100 ( hereinafter called bl100 ) , fancc - fance - fancf ( cef ) , and fanca - fancg - faap20 ( ag20 ) ( huang et  al . , 2014 , rajendra et  al . , 2014 ) . while the ag20 and cef play loosely defined ancillary roles ( wang and smogorzewska , 2015 )
, the bl100 complex is a three - member ubiquitin ligase , with fancl as the minimal unit required for ubiquitination ( huang et  al . , 2014 ,
fancl is an e3 ligase containing a ring domain that is essential for the interaction with the e2 enzyme , ube2t / fanct , and for the mono - ubiquitination of the fanci and fancd2 molecular targets ( cole et  al . , 2010 , hodson et  al . , 2011 , hodson et  al . , 2014 , machida et  al
although fancl is active in isolation , its catalytic function is greatly stimulated by the interaction with fancb and faap100 ( huang et  al .
an activating conformational switch in the fancl catalytic center , or whether fancb and faap100 play an architectural role important for mono - ubiquitination , is unknown .
multiple other architectural aspects of the mono - ubiquitination of fanci and fancd2 likewise remain poorly understood .
first , crystallographic studies on mouse proteins indicate that the fanci - fancd2 ( id2 ) hetero - dimer forms a pseudo-2-fold symmetric arrangement . however , the mono - ubiquitination sites map at the dimerization interface within the id2 complex ( joo et  al . , 2011 ) , suggesting that a reconfiguration of this interface is needed at some point during the mono - ubiquitination reaction to allow for access of the e3 ligase assembly .
second , whether a stable id2 complex is recruited to the dna damage site prior to mono - ubiquitination is unclear , although recent cryoelectron microscopy ( cryo - em ) studies on the unmodified human proteins support this notion ( liang et  al . , 2016 ) .
remarkably the symmetric hetero - dimer captured by crystallography can not be recognized in the electron microscopy ( em ) structure , suggesting that the id2 complex might visit different conformational states throughout the ubiquitination reaction .
finally , although both members of the id2 dimer need to be mono - ubiquitinated for activation of the icl repair pathway ( sims et  al . , 2007 , smogorzewska et  al . , 2007 , timmers et  al . , 2001 ) ,
so far , biochemical reconstitution has mainly focused on the minimal ability of the bl100 e3 ligase to mono - ubiquitinate fancd2 , with little to no  fanci mono - ubiquitination observed ( rajendra et  al.,2014 ) .
how equivalent mono - ubiquitination of both fanci and fancd2 is achieved and whether any of the ancillary proteins in the fa core complex are involved in this process remain unresolved questions . to address these issues ,
we have used em , combined with crosslinking and mass spectrometry ( xl / ms ) , to analyze the architecture of the bl100 complex and its substrate id2 , either in isolation or with other components of the fa core complex .
we found that bl100 forms a symmetric dimer of trimers , inviting a molecular model for the equivalent ubiquitination of the dimeric id2 assembly .
the cef component of the fa core complex binds to the two extremities in the bl100 dimer , probably acting as a molecular matchmaker that mediates the bl100 and id2 interaction .
we also uncovered an unexpected structural function for cef , which stabilizes the dimerization interface of the id2 complex .
we postulate that this function might have a direct role in the efficient mono - ubiquitination of the id2 hetero - dimeric substrate .
our findings provide key insights into the structural basis for the mono - ubiquitination of the id2 complex , the defining step in the activation of the fa / brca icl repair pathway .
although the fancl subunit of the core complex is an active e3 ligase in isolation ( alpi et  al . , 2008 ) , its mono - ubiquitination function is stimulated by 6-fold in the presence of fancb and faap100 ( rajendra et  al . , 2014 ) .
to understand whether the b100 association provides a structural framework for efficient mono - ubiquitination , or whether it causes a conformational change in fancl that stimulates its activity , we have expressed and purified human bl100 from insect cells .
several steps of purification yielded a pure trimeric complex that eluted with a sharp peak under gel filtration and with an apparent molecular weight of 440  kda ( figures 1a and s1 ) .
em imaging confirmed that our preparation is highly homogeneous and monodisperse ( figures 1b and 1c ) .
two - dimensional classification of negative stain data revealed an elongated bow - tie structure containing apparent 2-fold symmetry ( figure  1d ) .
the particle consists of a compact center and two globular modules , mapping at the two extremities of the assembly .
cryo - em analysis yielded classes that were highly reminiscent of those obtained after negative stain imaging ; however , no significant improvement in resolution could be detected , hinting at an inherent flexible nature in the bl100 complex ( figure  1e ) . to test our hypothesis that bl100 is mobile and
can adopt different structural states , we have performed focused classification on the central core of the structure , followed by classification on the whole particle without further alignment .
this approach revealed that the globular domains at the two poles in the structure move as rigid bodies , either tilting or stretching , relative to the central module ( figure  s1 ; movie s1 ) . despite this flexibility
, the apparent 2-fold symmetry is maintained in the central core and at the two poles in the structure . from the data shown so far ,
it remains unclear whether bl100 is , indeed , a 2-fold symmetric assembly or , rather , contains a pseudo-2-fold symmetry axis , similar to id2 ( figure  1f ) . to address this issue ,
we have repeated the two - dimensional analysis , using a fancl derivative that contains maltose - binding protein ( mbp ) fused to the n terminus ( figures 2a and s2 ) .
mbp tags provide an electron - dense feature that can be averaged in negative stain em , and they serve as pointers to localize individual proteins in a macromolecular assembly ( chen et  al . , 2008 ) .
we reasoned that a bona fide 2-fold symmetric complex should show two ( fancl - fused ) mbp densities that are dyad related .
indeed , as evident from two - dimensional class averages , b-(mbp)l100 is decorated with two symmetry - related mbp densities , establishing that the three factors assemble into a homo - dimer ( b : l:100  = 2:2:2 stoichiometry , with an expected molecular weight of 468  kda , coherent with our analytical gel filtration profile ) ( figures 2b and 2c ) .
a dimeric nature of the fa core complex was unexpected and invites a mechanistic model for the id2 mono - ubiquitination reaction that is further detailed in the discussion section .
previous reports indicate that ring domains can dimerize ( brzovic et  al . , 2001 ,
2011 ) , and our mbp analysis shows that the ring - containing fancl subunit maps in proximity of the dimerization interface of bl100 ( figures 2b and 2c ) . to test whether the fa core complex dimerization requires fancl ,
we have expressed and purified a subunit dropout assembly that only contains fancb and faap100 for single - particle analysis ( figures 2d and s3 ) . in the absence of fancl ,
the dimerization interface remains intact , while the globular poles in the assembly become markedly flexible ( figures 2e and s3 ; movie s2 ) , revealing a structural role for fancl in stabilizing the bl100 assembly ( figure  2f ) , in line with previous reports ( alpi et  al .
, we have attempted to purify the isolated fancb subunit , but we found that this factor is insoluble when expressed in the absence of faap100 ( data not shown ) .
conversely , the faap100 subunit is soluble and monodisperse in the absence of fancb , according to analytical gel filtration and single - particle em ( figures 2 g and s3 ) .
comparison of faap100 with the b100 two - dimensional class averages , centered on the flexible globular pole component , allows us to recognize faap100 as the element mapping at the extremities of the b100 particle .
no resemblance was detected between the isolated faap100 and the dimerization core in the assembly ( figures 2h and s3 ) . from these data , we conclude that fancb and faap100 provide a dimeric scaffold to position fancl on opposite poles in the protein assembly ( figure  2i ) .
this suggests that efficient substrate ubiquitination requires a 2-fold symmetric arrangement in the fa core complex .
furthermore , our subunit dropout experiments are incompatible with a homo - dimerization role for either fancl or faap100 , leaving the fancb subunit as the sole candidate for the dimerization element of the bl100 complex .
analytical biochemistry combined with single - particle em analysis allowed us to propose a model for the molecular architecture of a dimeric bl100 assembly .
difference mapping suggests that fancb is located at the homo - dimerization interface ; however , we failed in our attempts to directly show that the isolated fancb can self - associate .
therefore , we turned to xl / ms analysis , a powerful tool to study the architecture of macromolecular assemblies , especially when combined with structural em ( bui et  al . , 2013 ) ( figure  3a ; table s1 ) .
we have used an amino - reactive bi - functional crosslinker ( bs3 ) to covalently bridge between surface - exposed primary amines that map within a distance range of 2630   between c of lysine residues .
samples were crosslinked and trypsinized , enriched for larger crosslinked peptides through gel filtration , and analyzed by nanolc - ms ( nano - liquid chromatography - mass spectrometry ) using an ltq orbitrap velos .
we first validated the xl / ms data by comparing our results with the available crystal structure of the human fancl central domain .
all detected crosslinks in fancl exist within solvent - exposed lysines that are spaced within a range compatible with the dimensions of the crosslinker spacing arm ( figure  3b ) .
this control provided us with confidence in the interaction network built for the rest of the protein assembly .
remarkably , fancb contains , by far , the highest frequency of intra - molecular crosslinks , followed by fancl ( figures 3a and 3c ) .
possible explanations for this deficiency include the unusually low number of lysines in this subunit ( 1.9% of the sequence , with 5.7% being the average in human proteins ) , their location in large peptides lacking trypsin - cleavage sites , or the absence of proximal lysines in the tertiary structure of faap100 .
instead , the majority of crosslinked lysines observed for faap100 were involved in inter - molecular crosslinks with fancb .
altogether , these data provide additional support to the notion that fancb maps at the homo - dimerization interface in the core complex ( figures 2 and 3c ) .
we note that one of the intra - fancb crosslinks detected involves surface - exposed lysines ( in position 35 and 39 ) , which flank a known fa - associated mutation ( l37s ) . given the immediate proximity of lys35 and lys39 , leu37 is likely solvent exposed and could serve a role in interacting with other fa proteins .
further confidence in our xl / ms results is derived from the striking match between the inter - molecular crosslinks and our biochemical / em analysis of the bl100 assembly .
for example , four crosslinks connect faap100 with fancb , in accord with our finding that fancb - faap100 can exist as an isolated assembly ( figures 3a and 3c ) .
fancl and fancb are connected by five crosslinks , which agrees with the observation that the mbp n - terminally fused to fancl maps in close proximity to the central fancb homo - dimer ( figure  2b ) .
conversely , fancl and faap100 are not crosslinked , in agreement with our findings that ( 1 ) co - expression of fancl and faap100 does not yield a l100 hetero - dimer ( data not shown ) and that ( 2 ) the two subunits only appear loosely associated in the globular pole in the bl100 particle ( figure  2i ) . in conclusion , xl / ms analysis , combined with our single - particle em , provides a compelling architectural description of the dimeric bl100 catalytic assembly of the fa core complex .
we have , so far , established that the bl100 catalytic assembly of the fa core complex forms a homo - dimer .
whether this arrangement extends to the rest of the core complex , or whether the full assembly contains a symmetry mismatch element , remains to be established .
therefore , we decided to purify the cef assembly and image it in isolation ( as an mbp - fancc fusion derivative to increase the overall molecular mass of the complex ) ( figures 4a and s4 ) .
( mbp)cef appears as an electron - dense , tri - lobed entity with no detectable 2-fold symmetry component ( figures 4b and s4 ) .
we have then reconstituted a bl100-(mbp)cef co - assembly ( figures 4c and s4 ) and found that particles maintain the characteristic dimeric architecture of bl100 and are decorated with either one or two cef additional densities , connected to the globular poles in the bow - tie structure ( figures 4d and s4 ) .
therefore , binding of two copies of cef to the bl100 complex is physically possible , indicating that the bl100 dimer acts as an organizing center for other core complex components .
whether two copies of ag20 are found in the full fa core complex remains to be established and will be the focus of future studies . in the bl100-cef complex , cef projects outward from the bl100 dimerization center and appears perfectly poised to latch onto the outer perimeter of the id2 mono - ubiquitination substrate ( figure  4d ) .
a direct contact between fance and fancd2 has been previously reported ( gordon et  al . , 2005 , pace et  al . , 2002 ,
2014 ) , and in an accompanying study , we show that human cef can bind both fanci and fancd2 directly ( van twest et  al . , 2017 ) .
reconstituting a cef - id2 assembly could , therefore , provide key insights into the mechanism of the fanci - fancd2 mono - ubiquitination by the fa core complex .
xenopus laevis id2 can be expressed and purified to homogeneity and efficiently processed in the mono - ubiquitination reaction by the human fa core complex ( van twest et  al .
to gain mechanistic insights into id2 mono - ubiquitination by the human fa core complex , we chose to produce xenopus id2 for single - particle em studies ( figure  s5 ) . in agreement with recently published work ( liang et  al . ,
2016 ) , we observed that id2 forms an asymmetric , flexible , dimeric complex , which resists averaging and high - resolution structure determination ( figure  s5 ) .
we hypothesized that cef binding might stabilize the id2 assembly , and we co - expressed the five proteins in the attempt to purify an id2-cef complex by affinity purification via a flag - tagged fanci subunit .
this purification strategy , including stringent ion exchange and size exclusion chromatography ( sec ) steps , yielded an apparently stoichiometric id2 assembly , according to coomassie - stained sds - page gel analysis , but only trace amounts of the cef assembly ( figure  s6 ) .
as expected from inspection of the sds - page gel , the vast majority of id2 particles show no sign of cef co - complex , and no clear evidence for cef association was detected by inspection of the two - dimensional classes . to our surprise , however , id2 particles from the cef co - expression experiments yielded neatly averaging two - dimensional classes ( figure  s6 ) .
a pseudo-2-fold symmetric end - on view can be recognized in the assembly and perfectly matches the two - dimensional projections of an electron density map generated from the pdb coordinates of the mouse id2 crystal structure ( pdb : 3s4w ) ( joo et  al . , 2011 )
, we repeated the co - expression experiment using a different cef derivative , containing a strep ii tag on fancc ( figure  5a ) .
single - particle analysis on the reconstituted complex confirms our initial observations ( figures 5b5d and s6 ) . to obtain a three - dimensional structure of the stabilized id2 assembly
, we generated an initial model using eman2 , followed by three - dimensional classification , and auto - refined the most populated three - dimensional class to 18.3   resolution using relion ( figure  6b ; figure  s7 ) .
rigid - body docking of the mouse atomic structure into the xenopus id2 map results in an excellent fit , with a striking match at the hetero - dimerization interface ( figure  6b ) .
conversely , minor adjustments would be needed in the c - terminal region of the two protomers to perfectly fit the atomic structure into the em density ( although we chose not to perform these local adjustments , given the limited resolution of our three - dimensional em data ) .
coherent with a more flexible nature of the fanci and fancd2 carboxy - terminal modules , analysis of complex dynamics based on two - dimensional class averages of end - on views indicates that the peripheral lobes in the id2 dimer are flexible ( movie s3 ) . in summary
, our characterization of the id2-cef interaction reveals that the two protein assemblies do not form a stoichiometric complex , at least in the experimental conditions tested .
remarkably , however , cef co - expression alters the configuration of the id2 dimer , stabilizing the dimerization interface and locking the assembly in a pseudo-2-fold symmetric structure ( figure  5d ) .
this unexpected observation has key implications for the mechanism of id2 mono - ubiquitination and suggests a role for the cef assembly of the fa core complex .
because the two strands of dna are antiparallel ( watson and crick , 1953 ) , the factors that spatially organize , cut , or open the double helix are often arranged as symmetric dimers .
for example , a dyad element can be found in the protein core of the nucleosome ( the building block of chromatin in eukaryotic cells ) ( richmond et  al . , 1984 ) , in dna integrases ( which concertedly cut the two dna strands to insert foreign dna ) ( engelman and cherepanov , 2014 ) , or in dna helicases that unwind the double helix ( li et  al . , 2015 ) .
this 2-fold symmetric architecture can then be propagated to a subsequent set of proteins that physically interact with the dna - binding dimer .
a paradigm for this concept is found in the eukaryotic origin of dna replication , where two replisomes are built symmetrically around a dimer of ring - shaped helicases ( araki , 2016 , costa et  al . , 2014 , deegan et  al . , 2016 ) by a set of regulatory and chaperone proteins that are , in turn , symmetric dimers ( itou et  al . , 2015 ) .
in our study , we report one additional example of how a 2-fold symmetric enzyme modifies a dimeric dna interactor . in this case
, a homo - dimeric regulator protein acts symmetrically on a hetero - dimeric assembly . in the fa / brca icl repair pathway
, the fanci - fancd2 dimer binds the double helix at the damaged site and acts as a landing platform that recruits a set of dna - repair proteins for icl processing ( ceccaldi et  al . , 2016 ) .
fanci and fancd2 are structural homologs that can arrange in a pseudo-2-fold - symmetric assembly ( joo et  al . , 2011 ) .
recruitment of the dna - repair machinery requires mono - ubiquitination of both factors ( smogorzewska et  al . , 2007 ) .
this function is provided by the fa core complex , an enzyme assembly whose architecture was previously unknown ( ceccaldi et  al . , 2016 , walden and deans , 2014 ) . here
, we showed that the trimeric center of the fa core complex ( bl100 ) exists as a symmetric homo - dimer ( figure  2c ) .
fancb and faap100 play an architectural role , orienting the two e3 ligases in an ideal position to symmetrically target the fanci and fancd2 ubiquitination sites ( figure  2i ) . in particular
we note that fancb is the only protomer in the bl100 catalytic module that contains disease - associated missense mutations , emphasizing the key role of this factor in the assembly of a functional fa core complex ( chandrasekharappa et  al . , 2013 )
inspection of em two - dimensional class averages reveals that the bl100 particle is dynamic , with the two catalytic poles of the complex free to  tilt and stretch with respect to the fixed fancb dimerization center ( figure  s1 ; movie s1 ) .
remarkably , this movement occurs symmetrically across the particle ( when the upper lobe moves to  the left , the lower lobe moves to the right , and vice versa ) .
if the catalytic subunit fancl is omitted , however , the two external lobes in the particle move independently of each other ( figure  s3 ; movie s2 ) .
although the functional significance of this concerted movement is unclear , it is evident that each of the two catalytic subunits in the assembly must be able to sense the state of the symmetry mate in the assembly , and this allosteric communication must be mediated by the fancb dimerization center . in summary ,
our study provides the first insights into the mechanics of this dimeric e3 ligase super - assembly and starts to explain how the fancl mono - ubiquitination activity can be stimulated by the fancb and faap100 architectural factors ( rajendra et  al . , 2014 ) .
the bl100 assembly also functions as the organizing center of the fa core complex , providing a dimerization scaffold for the recruitment of a set of two cef trimeric assemblies ( figures 4d and s4 ) .
the fance subunit of cef has been previously reported to interact with the id2 assembly ( gordon et  al . , 2005 , pace et  al . , 2002 ,
polito et  al . , 2014 ) , and our structural data suggest that cef might act as a molecular matchmaker to bridge between the id2 mono - ubiquitination substrate and the bl100 e3 ligase machinery .
previous structural studies suggest that the id2 dimer might exist in different configurations during the ubiquitination reaction .
for example , crystallization of the murine complex captured a  symmetric , compact state of the id2 assembly , with the ubiquitination sites mapping at the dimerization interface , probably impairing access to the ubiquitination machinery ( joo et  al . , 2011 ) .
conversely , a cryo - em study of the human id2 shows that the complex is more flexible ( poorly averaged ) in solution ( liang et  al . , 2016 ) .
in agreement with these recent findings , we show that the intact xenopus id2 complex expressed in isolation can not be captured in discrete conformational states ( two - dimensional images do not average ) ( figure  s5 ) . in contrast , the co - expression of cef with id2 appears to stabilize a symmetric state of id2 , which is highly reminiscent of the form captured in the crystal structure ( figures 5c and 6 ; movie s3 ) .
this hints at a possible role for cef in manipulating the id2 substrate to facilitate symmetric mono - ubiquitination of the two related protomers ( figure  7 ) .
biochemical analysis of the reconstituted id2 mono - ubiquitination reaction by the fa core complex supports our suggestion that the cef assembly acts as a nexus between the bl100 enzyme and the id2 substrate while also reconfiguring the id2 structure ( van twest et  al . , 2017 ) .
whereas the isolated bl100 assembly solely ubiquitinates the fancd2 subunit , the presence of cef significantly stimulates fanci mono - ubiquitination , necessary for triggering initiation of the fa / brca icl repair pathway .
how bl100-cef and id2 interact and influence one another remains to be explained at a molecular level .
we note that a disease - associated mutation on fance ( r371w ) , predicted to destabilize the fance fold , abrogates the interaction with fancd2 in a yeast two - hybrid study . the same arg - to - trp point mutation on fancd2 ( r302w ) , mapping within the outer perimeter of the n - terminal id2 dimerization module , likewise abrogates the fance - d2 interaction ( joo et  al . , 2011 ,
such elements likely play an important role in the cef - mediated stabilization of the id2 substrate during the mono - ubiquitination reaction catalyzed by the homo - dimeric bl100 catalytic module .
notably , in addition to interacting with fance , the same r302 element in fancd2 has been previously implicated in mediating a histone h3 contact ( sato et  al . ,
how the id2 mono - ubiquitination reaction occurs in the context of chromatinized dna is an exciting frontier topic .
several outstanding questions will need to be addressed to understand the mechanism of activation of the fa / brca icl repair pathway .
for example : how do disease mutations affect the structure and function of the fa core complex ? what is the molecular mechanism for the formation of the fa core complex in cells ?
our discovery that the fa core complex is a symmetric dimer changes the way we think about these problems .
baculoviruses were constructed as detailed in the supplemental information . for the expression of the fancb - fancl - faap100 complex and the isolated faap100 ,
1  l of sf9 cells at a titer of 2.0   10 cells per milliliter were suspended in 4  l spinner flasks ( corning ) and infected with baculovirus at an moi of 2 for 72  hr at 27c .
cells were collected post - infection by centrifugation ( 5,000  rpm , 4c , 15  min ) , washed in pbs buffer supplemented with 5  mm mgcl2 , and resuspended in lysis buffer ( 25  mm hepes naoh [ ph 7.6 ] , 15  mm nacl , 0.02% tween 20 , 10% glycerol , 1  mm edta , 1  mm egta , 2  mm mgcl2 , 2  mm 2-bme , 0.4  mm pmsf , and roche protease inhibitor cocktail ) .
the suspension was lysed by dounce homogenization ( 40 strokes , tight pestle ) , and nacl was added to a final concentration of 250  mm .
the lysate was cleared by centrifugation ( 15000  rpm , 4c , 35  min ) , and the supernatant was incubated ( 3  hr , 4c ) with anti - flag m2 affinity resin ( sigma aldrich ) pre - equilibrated with flag washing buffer ( 25  mm hepes naoh [ ph 8.0 ] , 200  mm nacl , 0.02% tween 20 , 10% glycerol , 1  mm edta , 1  mm egta , and roche protease inhibitor cocktail ) . after wash , the sample was eluted with flag elution buffer ( 25  mm hepes naoh [ ph 8.0 ] , 100  mm nacl , 0.02% tween 20 , 5% glycerol , 1  mm edta , 1  mm egta , 0.3  mg / ml flag peptide , and roche protease inhibitor cocktail ) .
elution fractions were combined and loaded onto a mono q 5/50 gl for ion - exchange chromatography ( ge healthcare ) .
the column was washed with q - wash buffer ( 25  mm hepes naoh [ ph 8.0 ] , 150  mm nacl , 5% glycerol , 1  mm edta , 1  mm egta , 0.5  mm tcep , and roche protease inhibitor cocktail ) and eluted over a 150-mm to 650-mm nacl linear gradient in the same buffer . the peak fraction from cation - exchange chromatography
was loaded onto a superose 6 pc 3.2/30 column ( ge healthcare ) operating on an ktamicro hplc ( high - performance liquid chromatography ) system ( ge healthcare ) and was equilibrated in 25  mm hepes naoh ( ph 8.0 ) , 250  mm nacl , and 1  mm tcep . the proteins were separated by sds - page , using 4%12% bis - tris gels ( invitrogen ) running for 50  min at 150  v in mops buffer or using 3%8% tris - acetate gels ( invitrogen ) running for 60  min at 150  v in tris - acetate buffer .
protein concentration was determined by measuring absorbance at 280  nm ( a280 nm ) using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies ) .
expression of the fancb - faap100 and fancb-(mbp)fancl - faap100 complexes were performed using the fancb - fancl - faap100 protocol ; however , infection was performed using an moi of 1 for 72  hr at 27c .
freshly purified samples ( bl100 at 1  mg / ml , b100 at 0.25  mg / ml , and faap100 at 1.5 
mg / ml ) were immediately used for negative - stain em or cryo - em experiments .
1  l of sf9 cells at a titer of 1.0   10 cells per milliliter was suspended in 4-l spinner flasks ( corning ) and infected with baculovirus at an moi of 1 for 72  hr at 27c .
cells were collected post - infection by centrifugation ( 5,000  rpm , 4c , 15  min ) , washed in pbs buffer supplemented with 5  mm mgcl2 , and resuspended in lysis buffer ( 25  mm hepes naoh [ ph 7.6 ] , 15  mm nacl , 0.02% tween 20 , 10% glycerol , 1  mm edta , 1  mm egta , 2  mm mgcl2 , 15  mm 2-bme , 0.6  mm pmsf , and roche protease inhibitor cocktail ) .
the suspension was lysed by dounce homogenization ( 40  strokes , tight pestle ) , and nacl was added to a final concentration of  200  mm .
the lysate was cleared by centrifugation ( 15,000  rpm , 4c , 35  min ) , and the supernatant was incubated ( 2  hr , 4c ) with amylose resin ( new england biolabs ) pre - equilibrated with amylose wash buffer ( 25  mm hepes naoh [ ph 8.0 ] , 150  mm nacl , 0.02% tween 20 , 10% glycerol , 1  mm edta , 1  mm egta , 0.25  mm tcep , and roche protease inhibitor cocktail ) .
after extensive wash , the sample was eluted by ligand competition using the amylose wash buffer supplemented with 20  mm maltose ( sigma ) .
peak fractions were pooled and loaded onto a mono q 5/50 gl for ion - exchange chromatography ( ge healthcare ) .
the column was washed with buffer q - a ( 50  mm hepes naoh [ ph 8.0 ] , 150  mm nacl , 5% glycerol , 1  mm edta , and 0.5  mm tcep ) and eluted over a linear salt gradient with buffer q - b ( 50  mm hepes naoh [ ph 8.0 ] , 1  m nacl , 5% glycerol , 1  mm edta , and 0.5  mm tcep ) .
peak fraction was successively loaded onto a superose 6 pc 3.2/30 column ( ge healthcare ) operating on an ktamicro hplc system ( ge healthcare ) and separated by sec in 25  mm hepes naoh ( ph 8.0 ) , 100  mm nacl , and 2  mm dtt .
sample concentration was measured by a280 nm using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies ) and the ( mbp)cef theoretical extinction coefficient .
freshly purified ( mbp)cef sample at a final concentration of 0.5  mg / ml was immediately used for preparation of negative - stain em grids or reconstitution with freshly purified bl100 sample .
briefly , 1  l of sf9 cells at a titer of 2.0   10 cells per milliliter was suspended in 4-l spinner flasks ( corning ) and co - infected with the two baculoviruses , each at moi of 1 , for 72  hr at 27c . following the co - infection ,
cells were collected by centrifugation ( 5,000  rpm , 4c , 15  min ) and washed in pbs buffer supplemented with 5  mm mgcl2 .
cells were resuspended in lysis buffer ( 25  mm hepes naoh [ ph 7.6 ] , 15  mm nacl , 0.02% tween 20 , 10% glycerol , 1  mm edta , 1  mm egta , 2  mm mgcl2 , 2  mm 2-bme , 0.4  mm pmsf , and roche protease inhibitor cocktail ) and lysed by dounce homogenization ( 40  strokes , tight pestle ) . to preserve complex integrity ,
the lysate was added with nacl to a final concentration of 150  mm and cleared by centrifugation ( 15,000  rpm , 4c , 35  min ) .
the supernatant was collected and incubated ( 3  hr , 4c ) with anti - flag m2 affinity resin ( sigma - aldrich ) pre - equilibrated in flag washing buffer ( 25  mm hepes naoh [ ph 8.0 ] , 120  mm nacl , 0.02% tween 20 , 10% glycerol , 1  mm edta , 1  mm egta , and roche protease inhibitor cocktail ) .
after extensive wash , the proteins were eluted by incubation of the resin in flag elution buffer ( 25  mm hepes naoh [ ph 8.0 ] , 120  mm nacl , 1  mm edta , 0.3  mg / ml flag peptide , and roche protease inhibitor cocktail ) .
peak fractions were combined and loaded onto a mono q 5/50 gl(ge healthcare ) column running in buffer q - a ( 25  mm hepes naoh [ ph  8.0 ] , 100  mm nacl , 5% glycerol , 1  mm edta , and 0.25  mm tcep ) .
elution of the sample was performed over a linear salt gradient with buffer q - b ( 25  mm hepes naoh ph 8.0 ] , 750  mm nacl , 5% glycerol , 1  mm edta , and 0.5  mm tcep ) . to increase sample homogeneity , the complex was further purified by pull - down of the strep - tag ii on fancd2 .
peak fraction from ion - exchange chromatography was incubated for 2  hr at 4c with strep - tactin superflow plus resin ( qiagen ) .
the resin was washed in strep - wash buffer ( 25  mm hepes naoh [ ph 8.0 ] , 120  mm nacl , 10% glycerol , 1  mm edta and 0.25  mm tcep ) and eluted by incubation with strep - wash buffer supplemented with 25  mm desthiobiotin .
fractions containing stoichiometric i - d2 complex were pooled and concentrated on amicon ultra mwco 10k centrifugal filters ( millipore ) .
concentrated sample was separated by size exclusion on a superdex 200 pc 3.2/30 column ( ge healthcare ) operating on an ktamicro hplc system ( ge healthcare ) running in 25  mm hepes naoh ( ph 8.0 ) , 120  mm nacl , and 1  mm tcep .
elution peak was analyzed by sds - page followed by instant blue safestain staining ( expedeon ) .
freshly purified id2 sample at a final concentration of 0.25  mg / ml was immediately used for preparation of negative - stain em grids .
co - expression of fanci - fancd2-fancc - fance - fancf was obtained by infecting sf9 cells with baculoviruses encoding for fanci , fancd2 , and either mbp - tagged or strepii - tagged cef .
briefly , 1  l sf9 cells at a titer of 2.0   10 cells per milliliter were co - infected with baculoviruses encoding for flag - fanci , strepii - fancd2 , and ( mbp)cef ( or ( strepii)cef ) , each at a moi of 1 .
protein extraction , purification , and identification were carried out as previously described for the id2 complex . the id2-(strepii)cef sample purified from ion - exchange chromatography and
the id2-(mbp)cef sample purified from sec were used for preparation of em grids by negative stain immediately after purification .
the reconstitution of the fancb - fancl - faap100-(mbp)fancc - fance - fancf complex was performed by mixing equal volumes of a freshly purified 1-m solution of bl100 and a 3-m solution of cef ( resulting in a molar ratio of 1:3 ) and loaded on a 30k mwco slide - a - lyzer dialysis cassette ( thermo scientific ) .
the protein mix was first dialyzed for 1  hr at 4c in buffer 500 ( 25  mm tris - hcl [ ph 8.0 ] , 500  mm nacl , 5% glycerol , and 1  mm dtt ) , then for 2  hr in buffer 350 ( 25  mm tris - hcl [ ph 8.0 ] , 350  mm nacl , 5% glycerol , and 1  mm dtt ) , and finally overnight in buffer 100 ( 25  mm tris - hcl [ ph 8.0 ] , 100  mm nacl , 5% glycerol , and 1  mm dtt ) . the sample was recovered from the dialysis cassette and loaded onto a superose 6 pc 3.2/30 column ( ge healthcare ) operating on a ktamicro fplc ( fast protein liquid chromatography ) system ( ge healthcare ) equilibrated in 25  mm tris - hcl ( ph 8.0 ) , 100  mm nacl , and 2  mm dtt .
in control experiments , isolated dialyzed bl100 and ( mbp)cef complexes were also subject to sec as described for the reconstituted samples .
samples were analyzed by sds - page on 3%8% criterion xt tris - acetate  gels ( bio - rad ) followed by instant blue safestain staining ( expedeon ) .
a 2- to 6-nm - thick carbon layer was evaporated onto freshly cleaved mica sheets ( ems ) using a q150te carbon coater ( quorum technologies ) .
carbon was incubated overnight at 50c before floating on 600-mesh copper grids ( agar scientific ) or quantifoil r1.2/1.3 300-mesh copper grids .
prior to sample incubation , the carbon - coated grid was glow discharged for 30  s at 45 ma using a 100 glow discharger ( ems ) .
serial dilutions of each protein sample were performed to achieve optimal particle concentration on the em grid ( final concentrations ranged from 30 to 5  g / ml ) .
a 4-l drop of the freshly purified sample solution was applied onto the glow - discharged grid and incubated for 1  min .
approximately 2  l of sample solution was absorbed by gentle side blotting , and the grid was immediately stained with four subsequent applications onto 4 50-l drops of a 2% ( wt / vol ) uranyl formate solution with gentle stirring for 8 s. after staining , the grid was blotted dry and stored at room temperature prior to imaging .
particles of reconstituted bl100-(mbp)cef , isolated ( mbp)cef , isolated id2 , and id2 co - expressed with ( mbp)cef were imaged on a tecnai g2 spirit lab6 transmission electron microscope ( tem ; fei ) operating at 120 kev ( electron microscopy science technology platform , the francis crick institute ) .
images were manually acquired on a ultrascan 1000 2k  2k charge - coupled device ( ccd ) camera ( gatan ) at a nominal magnification of 30,000 ( corresponding to a pixel size of 3.45   at the specimen level ) .
particles of bl100 , b100 , b-(mbp)l100 , isolated faap100 , and id2 co - expressed with ( strepii)cef samples were imaged using a jem-2100 lab6 electron microscope ( jeol ) operated at 120 kv .
images were recorded on a ultrascan 4k  4k ccd camera ( gatan ) at  a nominal magnification of 42,000 corresponding to a pixel size of 2.73   at the specimen level . on both microscopes ,
images were collected in low - dose mode ( electron dose of 35 e /  ) and with defocus values in the range of 0.5  m to 2.0  m .
for the bl100 and faap100 samples , the micrographs were automatically recorded using the jeol automated data acquisition system ( jadas ) with same electron dose and defocus value as the manual collection .
a total of 567 images were collected for the bl100 sample , 250 images for the b100 sample , 412 images for the b-(mbp)l100 sample , 540 images for the faap100 sample , 277 images for the ( mbp)cef sample , 455 images for the  bl100-(mbp)cef sample,712 images for the id2 sample , 468 images for the id2-(mbp)cef sample , and 447 images for the id2-(strepii)cef sample .
ultrathin carbon - coated lacey 300-mesh copper grids ( ted pella ) were glow discharged for 30  s at 45 ma using a 100 glow discharger ( ems ) and mounted onto a vitrobot mark iv ( fei ) . a 4-l drop of freshly purified bl100 at a concentration of 20 
the sample was incubated on the grid for 1  min in 100% humidity at 21c .
after incubation , the grid was double - side blotted for 2.0  s and rapidly plunged in liquid ethane .
cryo - em data for the bl100 sample were collected on a titan krios tem ( fei ) operated at 300 kev and equipped with a back - thinned falcon ii ( fei ) direct electron detector at a nominal magnification of 47,000 , resulting in a pixel size of 1.77   at the specimen level .
a total of 2,798 images were recorded at defocus values in the range of 2.54.5  m and with a cumulative electron dose of 56 e /  distributed over 26 frames .
beam - induced motion was corrected on each image using the motioncorr program ( li et  al . , 2013 ) .
micrographs showing contamination drift , or out - of - range defocus values were discarded . for each dataset , particles were semi - automatically picked using the eman2 package , version 2.12 ( tang et  al . , 2007 ) .
from the negative - stain em data collection , a total of 63,543 individual particles were extracted from the bl100 dataset , 63,365 particles from the b100 dataset , 51,941 particles from the b-(mbp)l100 dataset , 177,106 particles from the faap100 dataset , 36,768 particles from bl100-(mbp)cef dataset , 47,898 particles from the ( mbp)cef dataset , 95,553 particles from the id2 dataset , 27,084 particles from the id2-(mbp)cef dataset , and 71,295 particles from the id2-(strepii)cef dataset . from the bl100 cryo - em dataset ,
the parameters of the contrast transfer function ( ctf ) were estimated on each micrograph using ctffind3 ( mindell and grigorieff , 2003 ) for negative - stain data and ctffind4 ( rohou and grigorieff , 2015 ) for cryo - em data .
reference - free two - dimensional alignment and averaging was performed using relion , version 1.4 ( scheres , 2012 ) . for three - dimensional reconstruction of the id2 complex co - expressed with ( strepii)cef ,
an initial three - dimensional volume was generated using the e2inimodel.py program in the eman2 package ( tang et  al . , 2007 ) .
the initial volume was used as a reference model for three - dimensional classification and refinement in relion 1.4 ( scheres , 2012 ) .
the atomic model of the fanci - fancd2 hetero - dimer ( pdb : 3s4w ) ( joo et  al . , 2011 ) was used for rigid - body fitting into the em map using ucsf ( university of california , san francisco ) chimera ( pettersen et  al . , 2004 ) .
the refined em map of the id2 hetero - dimer has been deposited in the three - dimensional - em database ( http://www.emdatabank.org ) with accession number : emd-3476 .
all chemicals were purchased from sigma at the highest grade possible , unless otherwise stated .
nano - lc - ms solvents and additives were purchased as lc - ms grade from fisher scientific .
the xl / ms workflow was conducted as previously described ( leitner et  al . , 2014 ) .
briefly , bl100 dimer , at a concentration of 0.5  mg / ml in 25  mm hepes ( ph 8) , 300  mm nacl , 10% glycerol , and 0.25  mm tris(2-carboxyethyl)phosphine ( tcep ) , was reacted with 5  mm bs3 ( using a 50:50 ratio of d0 and d4 ) for 30  min at 37c .
the reaction was quenched by adding 50  mm ammonium bicarbonate for 20  min at 37c and then dried to completion using vacuum centrifugation .
the sample was resuspended at a concentration of 1  mg / ml using 8  m urea , reduced with 2.5  mm tcep , and alkylated with 5  mm iodoacetamide .
the sample was diluted with 50  mm ammonium bicarbonate to reduce the urea concentration to 1 m , and trypsin ( promega ) was added at a 1:50 enzyme : substrate ratio and left to digest overnight at 37c .
the solution was then acidified , and peptides were purified using c18 in house solid - phase elution tips ( empore , 3 m ) . for size exclusion fractionation , the peptides were resuspended in sec buffer ( 70% h2o , 30% acetonitrile , and 0.1% trifluoroacetic acid ) , and chromatographically separated using an ktamicro ( ge healthcare ) with a superdex peptide pc 3.2/30 column ( ge healthcare ) and detected using 215  nm absorbance .
the flow rate was set to 50  l / min , and 100  l fractions were collected and dried to completion using vacuum centrifugation . for nano - lc - ms acquisition , the sec fractions were resuspended into 30  l of 0.1% trifluoroacetic acid in h2o , and 1  l was injected and chromatographically resolved using an ultimate 3000 rslcnano ( dionex ) with an easy - spray column ( 2-m particles , pepmap c18 , 100- pore size , 50  cm  75  m i d ) ( thermo scientific ) .
a flow rate of 0.25  l / min was used , starting at 98% mobile a ( 0.1% formic acid , 5% dmso , 95% h2o ) and 2% mobile b ( 0.1% formic acid , 5% dmso , 80% acetonitrile , 10% h2o ) . over 80 
min , mobile b was then increased to 40% followed by a further increase to 90% over 10  min .
spectra were acquired in real time using an ltq orbitrap velos mass spectrometer ( thermo scientific ) with a 400 to 2,000 m / z ( mass - to - charge ratio ) acquisition window .
the top ten most intense ions with a charge state of  + 3 or greater were then selected for tandem ms ( ms / ms ) using data - dependent acquisition and cid ( collision - induced dissociation ) fragmentation ( at 35% normalized collision energy ) . a dynamic exclusion list , with a repeat count of 1 , repeat duration of 20 s , exclusion list size of 100 , and exclusion duration of 30 s , was used to prevent repeat sampling .
the data were searched for bs3 mono - links ( + 156.079  da for water quench ,  + 155.096  da for ammonium quench ) , intra - links , and intra- and inter - protein crosslinks ( + 138.068 da ) on lysine residues , using isotopic pairing with a mass shift of  + 4.025  da for bs3 d4 ions , in addition to a fixed carbamidomethylation modification ( + 57.021 da ) and variable methionine oxidation ( + 15.994 da ) .
the data were searched using a 10-ppm mass tolerance for charge states  + 3 or above .
xprophet was then used , and peptides with a false discovery rate ( fdr ) below 5% were then manually assessed for adequate signal intensity and peak assignment .
, s.v.t . , and f.s . constructed all baculoviruses and performed preliminary protein preparations .
optimized purification protocols for structural studies , prepared all proteins and em grids , collected em data ( with l.r . ) , and performed all image analysis under the supervision of a.c .
 summaryactivation of the main dna interstrand crosslink repair pathway in higher eukaryotes requires mono - ubiquitination of fanci and fancd2 by fancl , the e3 ligase subunit of the fanconi anemia core complex . 
 fanci and fancd2 form a stable complex ; however , the molecular basis of their ubiquitination is ill defined . 
 fancd2 mono - ubiquitination by fancl is stimulated by the presence of the fancb and faap100 core complex components , through an unknown mechanism . 
 how fanci mono - ubiquitination is achieved remains unclear . here 
 , we use structural electron microscopy , combined with crosslink - coupled mass spectrometry , to find that fancb , fancl , and faap100 form a dimer of trimers , containing two fancl molecules that are ideally poised to target both fanci and fancd2 for mono - ubiquitination . the fancc - fance - fancf subunits bridge between fancb - fancl - faap100 and the fanci - fancd2 substrate . a transient interaction with fancc - fance - fancf 
 alters the fanci - fancd2 configuration , stabilizing the dimerization interface . 
 our data provide a model to explain how equivalent mono - ubiquitination of fanci and fancd2 occurs . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions