The FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer predisposition syndrome, is required for the repair of interstrand crosslinks (ICL) and related lesions 1 . The FA pathway is activated when a replication fork stalls at an ICL 2 , triggering the mono-ubiquitination of the ID complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion synthesis and homologous recombination, but its function was hitherto unknown 1 , 3 . Here, the 3.5 Å cryo-EM structure of mono-ubiquitinated ID (ID Ub ) bound to DNA reveals that it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. The structures, in conjunction with biochemical data, indicate the mono-ubiquitinated ID complex looses its preference for ICL and related branched DNA structures, becoming a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how mono-ubiquitination in general can induce an alternate structure with a new function.

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15The FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer 16 predisposition syndrome, is required for the repair of replication forks stalled at DNA 17 interstrand crosslinks (ICL) and related lesions 1 . The FA pathway is activated when two 18 replication forks converge onto an ICL 2 , triggering the mono-ubiquitination of the ID 19 complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion 20 synthesis and homologous recombination, but its function was hitherto unknown 1,3 . Here, 21 the 3.48 Å cryo-EM structure of mono-ubiquitinated ID (ID Ub ) bound to DNA reveals that 22 it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the 23 non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-24 ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure 25 through the ubiquitin of one protomer binding to the other protomer in a reciprocal 26 fashion. The structures, in conjunction with biochemical data, indicate the mono-27 ubiquitinated ID complex looses its preference for ICL and related branched DNA 28 structures, becoming a sliding DNA clamp that can coordinate the subsequent repair 29 reactions. Our findings also reveal how mono-ubiquitination in general can induce an 30 alternate structure with a new function. 31 32 FANCI and FANCD2 are paralogs that bind to DNA with preference for branched 33 structures including Holliday junction, overhang and replication fork DNA 4-7 . The previous 34 crystal structure of the mouse ID complex showed that it forms an open trough-like structure 35 with two basic grooves, one on each paralog 7 . A 7.8 Å crystallographic map of FANCI bound to 36 3splayed Y DNA confirmed that its basic groove is the site of dsDNA binding, and also identified 37 a likely single-stranded DNA binding region 7 . However, it has not been clear how these DNA 38 binding activities relate to the function of the ID complex in replication and ICL repair. The 39 mouse ID structure also showed the mono-ubiquitination sites are embedded inside the FANCI-40 FANCD2 interface 7 , but did not shed light on the function of mono-ubiquitination. 41 To address these questions, we first collected cryo-EM data on the human, full-length ID 42 complex bound to an ICL-containing DNA constructed by crosslinking two modified 43 oligonucleotides with a triazole moiety 8 (Fig. 1a). This ICL DNA mimics two replication forks 44 converging on an ICL, an event shown to activate the FA pathway 2 . The initial consensus 45 reconstruction with 231,943 particles extended to 3.40 Å, as determined by the gold-standard 46 fourier shell correlation (FSC) procedure 9 (Extended Data Figs 1a and b). The map showed that 47

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.