- EMDB-24862: CryoEM structure of modular PKS holo-Lsd14 bound to antibody frag... -
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データベース: EMDB / ID: EMD-24862
タイトル
CryoEM structure of modular PKS holo-Lsd14 bound to antibody fragment 1B2, stalled at the condensation step, consensus refinement
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試料
複合体: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM136258
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM138990
米国
National Science Foundation (NSF, United States)
DMR-1719875
米国
引用
ジャーナル: Science / 年: 2021 タイトル: Modular polyketide synthase contains two reaction chambers that operate asynchronously. 著者: Saket R Bagde / Irimpan I Mathews / J Christopher Fromme / Chu-Young Kim / 要旨: Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto ...Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo–electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.
名称: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate
要素
複合体: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate
名称: Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoate タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#3
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: Blot for 5 seconds before plunging in liquid ethane..
詳細
Size exclusion chromatography purified monodisperse holo-Lsd14-DD* + 1B2 complex was first incubated with KS substrate analog (2-Acetaminoethyl-thio-3-oxobutanoate) and TCEP at 30C for 20 minutes, followed by addition of NADP and methylmalonyl-CoA. The sample was further incubated at 30C for 15 minutes before applying to cryoEM grids.