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Transcription-coupled DNA repair removes bulky DNA lesions from the genome 1 , 2 and protects cells against ultraviolet (UV) irradiation 3 . Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4 CSA and UV-stimulated scaffold protein A (UVSSA) 3 . Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published 3 , 4 data, the structures provide a model for transcription–repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, EC TCR , uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4 CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4 CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA. The authors resolve the structure of five complexes containing RNA polymerase II and the CSA and CSB proteins, offering insight into how the repair of DNA lesions is coupled to transcription.

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.Cryo-EM structures of the S. cerevisiae condensin holo complex reveal that ATP binding triggers exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains, potentially leading to an interconversion of DNA-binding sites in the catalytic core of condensin that might form the basis of its DNA translocation and loop-extrusion activities.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria after synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry: pore-forming β-barrel protein Tom40 and four auxiliary α-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface attributed to multiple functionally important acidic patches. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the detailed molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly 1 . Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years 2 . Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin’s motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state 3 ; how titin and cMyBP-C may contribute to length-dependent activation 4 ; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease 5 , 6 . The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs. The intricate molecular architecture and interactions of the human cardiac myosin filament offer insights into cardiac physiology, disease and drug therapy.

The cullin–RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A–RBX1–DDB1–DDB2 complex (CRL4A DDB2 ) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A DBB2 is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A DDB2 and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN. Much of the intracellular protein degradation in eukaryotes is controlled by cullin–RING ubiquitin ligases (CRLs), a vast class of enzymes which are regulated by the COP9 signalosome (CSN); structural characterization of CSN–N8CRL4A complexes by cryo-electron microscopy reveals an induced-fit mechanism of CSN activation triggered only by catalytically activated CRLs without bound substrate, explaining how CSN acts as a global regulator of CRLs. Control of intracellular protein degradation Much of the intracellular protein degradation in eukaryotes is controlled by cullin–RING ubiquitin ligases (CRLs). The structure of these enzymes and their substrates vary greatly, yet all are regulated by a single complex — the COP9 signalosome (CSN). What enables CSN to be a master regulator of diverse CRLs? Nicolas Thomä and colleagues present biochemical data and cryo-electron microscopy of CSN–CRL4 complexes revealing an induced-fit mechanism that activates CSN only in the presence of a catalytically activated CRL not bound to a substrate. The authors identify both unique and less-specific CSN–CRL contacts.

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR) 1 – 5 . In the DNA damage responses, this post-translational modification occurs predominantly on serine residues 6 – 8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine 9 , 10 . Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks 2 , 11 . Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2–HPF1 bound to a nucleosome. This showed that PARP2–HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD + and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments 12 – 16 . The PARP2–HPF1 histone-modifying complex bridges two nucleosomes to align broken DNA ends for ligation, initiating conformational changes that activate PARP2 and enable DNA damage repair.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes 1 – 6 . After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails) 7 – 9 , whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails 10 – 12 . In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis 13 . Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins. Cryo-electron microscopy structures of the yeast 60S ribosomal subunit in complex with Vms1 provides insights into the roles of proteins in the ribosome-associated quality control pathway.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the “C-zone.” Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration 1 . Together with hexanucleotide-repeat expansion 2 , 3 , haploinsufficiency of C9orf72 contributes to neuronal dysfunction 4 – 6 . Here we determine the structure of the C9orf72–SMCR8–WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains 7 , and WDR41 is a β-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72–SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72–SMCR8–WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration. The cryo-electron microscopy structure of C9orf72–SMCR8–WDR41 suggests that this complex is a GTPase-activating protein for ARF-family small GTPases, which sheds light on the role of C9orf72 mutations in neuronal dysfunction.

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors. Multidrug efflux pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the efflux pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.