Cryo-EM structure of the Smc5/6 holo-complex

Hallett, Stephen T; Harry, Isabella Campbell; Schellenberger, Pascale; Zhou, Lihong; Cronin, Nora; Baxter, Jonathan; Etheridge, Thomas J.; Murray, Johanne M.; Oliver, Antony W.

doi:10.1101/2021.11.25.470006

Cited by 4 publications

(8 citation statements)

References 78 publications

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“…However, the impact of these high-quality models in structural biology goes far beyond the simple case of molecular replacement. Numerous examples have illustrated how these models can be docked into cryo-EM maps and used to interpret them, to assign side chains or to improve the quality of the built models (Gupta et al, 2021;Hallett et al, 2021;He et al, 2021;Peter et al, 2021;Tai et al, 2021;de la Pen ˜a et al, 2021). The iterative input of models grossly fitted into cryo-EM or crystallographic maps in AlphaFold can also be used to improve the resulting models generated by this program (Terwilliger et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report

Barbarin-Bocahu¹,

Graille²

2022

Acta Crystallogr D Struct Biol

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The breakthrough recently made in protein structure prediction by deep-learning programs such as AlphaFold and RoseTTAFold will certainly revolutionize biology over the coming decades. The scientific community is only starting to appreciate the various applications, benefits and limitations of these protein models. Yet, after the first thrills due to this revolution, it is important to evaluate the impact of the proposed models and their overall quality to avoid the misinterpretation or overinterpretation of these models by biologists. One of the first applications of these models is in solving the `phase problem' encountered in X-ray crystallography in calculating electron-density maps from diffraction data. Indeed, the most frequently used technique to derive electron-density maps is molecular replacement. As this technique relies on knowledge of the structure of a protein that shares strong structural similarity with the studied protein, the availability of high-accuracy models is then definitely critical for successful structure solution. After the collection of a 2.45 Å resolution data set, we struggled for two years in trying to solve the crystal structure of a protein involved in the nonsense-mediated mRNA decay pathway, an mRNA quality-control pathway dedicated to the elimination of eukaryotic mRNAs harboring premature stop codons. We used different methods (isomorphous replacement, anomalous diffraction and molecular replacement) to determine this structure, but all failed until we straightforwardly succeeded thanks to both AlphaFold and RoseTTAFold models. Here, we describe how these new models helped us to solve this structure and conclude that in our case the AlphaFold model largely outcompetes the other models. We also discuss the importance of search-model generation for successful molecular replacement.

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Section: Discussionmentioning

confidence: 99%

The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report

Barbarin-Bocahu¹,

Graille²

2022

Acta Crystallogr D Struct Biol

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“…Recent CLMS and EM data suggest that DNA-free Smc5/6 has a closed arm configuration containing head-proximal Nse1-3 ( 10 – 14 , 22 ). By contrast, the structure of DNA-bound Smc5/6 shows arm opening and Nse1-3 shifting to above the head regions, suggestive of major conformational changes.…”

Section: Resultsmentioning

confidence: 99%

“…DNA loop extrusion additionally requires arm bending at a region called the elbow, which is found in both cohesin and condensin ( 5 , 7 – 9 ). By contrast, a lack of arm bending in Smc5/6 was suggested by negative stain EM and cross-linking mass spectrometry (CLMS) data ( 10 – 14 ). Since Smc5/6 does not contain HAWK proteins nor shows arm-bending, it has remained unclear how Smc5/6 engages DNA to accomplish its multiple functions.…”

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confidence: 98%

Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes

Ser

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Significance The Smc5/6 complex plays multiple roles in DNA replication and repair. Its genome-protecting functions rely on its interaction with DNA; however, how this complex engages DNA is poorly understood. We report on a cryogenic electron microscopy structure of DNA-bound budding yeast Smc5/6 complex, revealing that its subunits form a clamp to encircle a double-helical DNA. We define the multi-subunit interactions forming the DNA clamp and the DNA binding sites distributed among subunits. We identify subunit transformations upon DNA capture and functional effects conferred by its multiple DNA contact sites. Our findings, in conjunction with studies on other structural maintenance of chromosomes (SMC) complexes, suggest a common SMC DNA-clamp mechanism with individual complex specific features that enable diverse genome organization and protection functions by SMC family complexes.

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“…All of them cause lethality when removed by gene disruption 9 . Smc5-6 and Nse4 together form the conserved ATPase core with an elongated shape that is characteristic for all SMC complexes [16][17][18][19][20][21] . Smc5 and Smc6 proteins harbor the canonical 'hinge' domains for heterotypic dimerization connected via ~35 nm long antiparallel coiled-coil 'arms' to globular ABC-type 'head' domains with highly conserved motifs for ATP-binding and hydrolysis [22][23][24] .…”

Section: Introductionmentioning

confidence: 99%

“…The ATP hydrolysis cycle involves major structural rearrangements in the SMC complex [36][37][38][39][40][41] . Three main conformations have been delineated for Smc5/6 (fig S1B) [16][17][18][19][20][21] :…”

Section: Introductionmentioning

confidence: 99%

DNA segment capture by Smc5/6 holo-complexes

Täschner

Gruber

2022

Preprint

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Three distinct SMC complexes facilitate chromosome folding and segregation in eukaryotes, presumably by DNA translocation and loop extrusion. How SMCs interact with DNA is however not well understood. Among the SMC complexes, Smc5/6 has dedicated roles in DNA repair and in preventing a lethal buildup of aberrant DNA junctions. Here, we describe the reconstitution of ATP-dependent topological DNA loading by Smc5/6 rings. By inserting cysteine residues at selected protein interfaces, we obtained covalently closed compartments upon chemical cross-linking. We show that two SMC sub-compartments and the kleisin compartment topologically entrap a plasmid molecule, but not the full SMC compartment. This is explained by a looped DNA segment inserting into the SMC compartment with the kleisin neck gate locking the loop in place when passing between the two DNA flanks and closing. This DNA segment capture strictly requires the Nse5/6 loader, which opens the neck gate prior to DNA passage. Similar segment capture events without gate opening may provide the power stroke for DNA translocation/loop extrusion in subsequent ATP hydrolysis cycles. Our biochemical experiments thus offer a unifying principle for SMC ATPase function in loading and translocation/extrusion, which is likely relevant to other members of the family of SMC proteins too.

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Cryo-EM structure of the Smc5/6 holo-complex

Cited by 4 publications

References 78 publications

The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report

The X-ray crystallography phase problem solved thanks to AlphaFold and RoseTTAFold models: a case-study report

Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes

DNA segment capture by Smc5/6 holo-complexes

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