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.
Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 ...and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the
form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (
) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding.
A folded conformation of MukBEF and cohesin Bürmann, Frank; Lee, Byung-Gil; Than, Thane ...
Nature structural & molecular biology,
03/2019, Letnik:
26, Številka:
3
Journal Article
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Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. ...They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head-kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.
Clamping of DNA shuts the condensin neck gate Lee, Byung-Gil; Rhodes, James; Löwe, Jan
Proceedings of the National Academy of Sciences - PNAS,
04/2022, Letnik:
119, Številka:
14
Journal Article
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SignificanceDNA needs to be compacted to fit into nuclei and during cell division, when dense chromatids are formed for their mechanical segregation, a process that depends on the protein complex ...condensin. It forms and enlarges loops in DNA through loop extrusion. Our work resolves the atomic structure of a DNA-bound state of condensin in which ATP has not been hydrolyzed. The DNA is clamped within a compartment that has been reported previously in other structural maintenance of chromosomes (SMC) complexes, including Rad50, cohesin, and MukBEF. With the caveat of important differences, it means that all SMC complexes cycle through at least some similar states and undergo similar conformational changes in their head modules, while hydrolyzing ATP and translocating DNA.
The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP‐dependent protease when coupled with Clp‐ATPases. ...Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP‐bound states. Cryo‐electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH‐lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self‐compartmentalizing protease.
Synopsis
Delineating the mechanisms of substrate engagement and product release is essential for understanding the role of the conserved ClpP protease in protein homeostasis and developing antibiotics. This study shows that the opening of ClpP side exit pores is coupled to a pH decrease caused by peptide accumulation in the proteolytic chamber.
Substrate hydrolysis by proteases lowers intra‐compartmental pH value by generating a large number of peptide products.
Crystal structures show BsClpP in complex with an unforeseen number of acyldepsipeptides (ADEPs).
Cryo‐EM structures of BsClpP at different pH values and in ADEP‐bound conditions reveal various structural features.
Side exit pores of BsClpP are gradually enlarged upon pH lowering.
Conformational changes linked to peptide‐mediated acidification suggest a mechanism for proteolytic product release by a key bacterial and mitochondrial ATP‐dependent Clp protease system.
To investigate the structural integrity of the superficial capillary plexuses (SCPs) and deep capillary plexuses (DCPs) using optical coherence tomography (OCT) angiography (OCTA) in patients with ...diabetic macular edema (DME) and its association with the response to anti–vascular endothelial growth factor (VEGF) treatment.
Retrospective, case-control study.
We included 51 DME eyes with a poor response to anti-VEGF agents and 32 age-matched DME eyes with a good response to anti-VEGF treatment, along with 20 fellow eyes without DME from the cases and controls.
The medical records, including OCTA and spectral-domain OCT (SD OCT), were reviewed and compared between the groups. En face OCTA images of the SCP and DCP were obtained for each eye. An anti-VEGF responder was defined by a reduction of more than 50 μm in central retinal thickness after 3 consecutive anti-VEGF treatments. A poor responder was defined by a reduction of less than 50 μm or an increase in central retinal thickness after 3 monthly injections.
We measured the vascular density and foveal avascular zone (FAZ) area and counted the number of microaneurysms in each layer. The SD OCT images were compared with OCTA findings.
Compared with non-DME eyes, DME eyes had a lower vascular density (P < 0.001) and larger FAZ area (P < 0.001) in the DCP and more microaneurysms (P < 0.001) in both layers. Although there was no significant difference in the SCP between anti-VEGF responders and poor responders, poor responders tended to show greater damage and more microaneurysms in the DCP (P < 0.001) and a larger FAZ area (P < 0.001). The topographic location of the disrupted synaptic portion of the outer plexiform layer (OPL) in SD OCT exactly corresponded to the nonflow area of the DCP in OCTA.
Compared with DME eyes that responded to anti-VEGF treatment, poor responders show significant damage to the integrity of the DCP, but not the SCP. The degree of OPL disruption in SD OCT corresponds well with the extent of DCP loss in DME eyes. The extent of DCP loss and the corresponding OPL disruption could be useful predictors of responsiveness to anti-VEGF treatment.
With the increase in research cases of the application of a convolutional neural network (CNN)-based object detection technology, studies on the light-weight CNN models that can be performed in real ...time on the edge-computing devices are also increasing. This paper proposed scalable convolutional blocks that can be easily designed CNN networks of You Only Look Once (YOLO) detector which have the balanced processing speed and accuracy of the target edge-computing devices considering different performances by exchanging the proposed blocks simply. The maximum number of kernels of the convolutional layer was determined through simple but intuitive speed comparison tests for three edge-computing devices to be considered. The scalable convolutional blocks were designed in consideration of the limited maximum number of kernels to detect objects in real time on these edge-computing devices. Three scalable and fast YOLO detectors (SF-YOLO) which designed using the proposed scalable convolutional blocks compared the processing speed and accuracy with several conventional light-weight YOLO detectors on the edge-computing devices. When compared with YOLOv3-tiny, SF-YOLO was seen to be 2 times faster than the previous processing speed but with the same accuracy as YOLOv3-tiny, and also, a 48% improved processing speed than the YOLOv3-tiny-PRN which is the processing speed improvement model. Also, even in the large SF-YOLO model that focuses on the accuracy performance, it achieved a 10% faster processing speed with better accuracy of 40.4% mAP@0.5 in the MS COCO dataset than YOLOv4-tiny model.
► We proposed multi-vessel collision risk assessment system based on fuzzy logic. ► The marine traffic management is becoming a hot topic due to increase in vessel number. ► The VTS crew has to ...calculate the collision risk manually to guide the ship captain. ► Our proposed system automatically calculates the associated risk among vessels from the VTS centre. ► The results derived from experiments prove the effectiveness of our solution.
Due to brisk industrial growth, the marine traffic has become an imperative subject in the open sea nowadays. The crew inside the vehicle traffic service (VTS) centre is facing challenging issues on account of continuous growth in vessel number. Currently, most of VTS centers’ are using the ARPA RADAR based conventional vehicle traffic management system and VTS staff has to carry out most of the things manually to guide the ship’s captain properly. Therefore, there is a strong impetus in the field of ocean engineering to develop a smart system which can take the data from RADAR and autonomously manipulate it, to calculate the degree of collision risk among all vessels from the VTS centre. Later on, the traffic management officer utilizes this information for intelligent decision making. In the past, several researchers have addressed this issue to facilities the VTS crew and captain of the ship but mostly, their research work was for academic purposes and could not get popularity because of extra manual workload. Our proposed vessel collision risk assessment system is an intelligent solution which is based on fuzzy inference system and has the ability to solve the said issues. We calculated the DCPA, TCPA, bearing and VCD among all vessels ships from the VTS centre by using conventional marine equipments and exploited the extracted information to calculate and display the degree of collision risk among all vessels. Furthermore, we developed the RADAR filtration algorithm which helps the VTS officer to gauge out the degree of collision risk around a particular ship. To authenticate the validity and to monitor the performance efficiency, we developed RADAR operated intelligent software which directly gets the required data from RADAR and displays the vessels list based on their degree of collision severity. The laboratory experiments confirm the validity of the proposed system.
In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped ...proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are 'clamped' in a sub-compartment created by Scc2's association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.
Cohesin organizes DNA into chromatids, regulates enhancer-promoter interactions, and confers sister chromatid cohesion. Its association with chromosomes is regulated by hook-shaped HEAT repeat ...proteins that bind Scc1, namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently replaces Pds5. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin’s ATPase activity and loading. Moreover, Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin’s initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2’s association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another in which Scc2 replaces Pds5 and stimulates ATP hydrolysis necessary for loading and translocation from loading sites.
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•Among Hawks, Scc2 is sufficient to activate cohesin’s ATPase in the presence of DNA•Scc1 mutants that compromise Scc2 binding reduce ATPase activity and cohesin loading•Scc2 gain-of-function mutations increase its association with chromosomal cohesin•Increased Scc2 association with chromosomal cohesin correlates with a decrease in Pds5
Cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another one in which Scc2 replaces Pds5 and stimulates ATP hydrolysis required for loading and translocation.
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