ジャーナル: Nat Commun / 年: 2019 タイトル: Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex. 著者: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe ...著者: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe Colletier / Peter Güntert / Adrien Favier / Guy Schoehn / Paul Schanda / Jerome Boisbouvier / 要旨: Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely ...Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.
BMRB: 34205Combined solid-state NMR, solution-state NMR and EM data for structure determination of the tetrahedral aminopeptidase TET2 from P. horikoshii
装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 293 K / 詳細: Blotting time 2s, force 1, drain time 0.
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Solution-ID
内容
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Label
溶媒系
solid
1
20mMTRIS, 50mMsodiumchloride, 100% H2O
protein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 2H15N and 13C at methyls of ILV.
2H15N_ILVCHD2
100% H2O
solid
5
20mMTRIS, 50mMsodiumchloride, 100% H2O
2H13C15N
100% H2O
solid
3
20mMTris, 50mMNaCl, 100% H2O
protein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 13C at all carbons of LKP residues