ジャーナル: Nucleic Acids Res / 年: 2022 タイトル: Cryo-EM structure of the Smc5/6 holo-complex. 著者: Stephen T Hallett / Isabella Campbell Harry / Pascale Schellenberger / Lihong Zhou / Nora B Cronin / Jonathan Baxter / Thomas J Etheridge / Johanne M Murray / Antony W Oliver / 要旨: The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also ...The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.
名称: Saccharomyces cerevisiae Smc5/6 holo-complex / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1 / 詳細: Map focussed at the head-end of the holo0complex
由来(天然)
生物種: Saccharomyces cerevisiae (パン酵母)
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実験情報
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構造解析
手法
クライオ電子顕微鏡法
解析
単粒子再構成法
試料の集合状態
particle
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試料調製
濃度
0.1 mg/mL
緩衝液
pH: 7.5 構成要素:
濃度
式
名称
20.0 mM
C8H18N2O4S
HEPES
50.0 mM
NaCl塩化ナトリウム
sodium chloride塩化ナトリウム
0.5 mM
C9H16ClO6P
TCEP
凍結
凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 283.15 K / 装置: LEICA EM GP 詳細: The grid was held in the chamber for a period of 10 seconds, before blotting for 2.5 to 4.5 seconds using the auto-sensor. Grids were then plunged into liquid ethane before being stored under ...詳細: The grid was held in the chamber for a period of 10 seconds, before blotting for 2.5 to 4.5 seconds using the auto-sensor. Grids were then plunged into liquid ethane before being stored under liquid nitrogen until data collection..
詳細
Fraction(s) eluting from a Superose 6 size exclusion chromatography column (Cytiva Life Sciences). Cross-linked with BS3 before grid preparation.