National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)
NIH DK 027044
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
NIH GM 110530
米国
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2022 タイトル: Munc13 structural transitions and oligomers that may choreograph successive stages in vesicle priming for neurotransmitter release. 著者: Kirill Grushin / R Venkat Kalyana Sundaram / Charles V Sindelar / James E Rothman / 要旨: How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin ...How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin assembly. The functional core of Munc13, consisting of C1-C2B-MUN-C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state. In the open conformation (state 1), Munc13C forms upright trimers that link the two bilayers, separating them by ∼21 nm. In the closed conformation, six copies of Munc13C interact to form a lateral hexamer elevated ∼14 nm above the bilayer. Open and closed conformations differ only by a rigid body rotation around a flexible hinge, which when performed cooperatively assembles Munc13 into a lateral hexamer (state 2) in which the key SNARE assembly-activating site of Munc13 is autoinhibited by its neighbor. We propose that each Munc13 in the lateral hexamer ultimately assembles a single SNAREpin, explaining how only and exactly six SNARE complexes are templated. We suggest that state 1 and state 2 may represent two successive states in the synaptic vesicle supply chain leading to "primed" ready-release vesicles in which SNAREpins are clamped and ready to release (state 3).
想定した対称性 - 点群: C6 (6回回転対称) / 解像度のタイプ: BY AUTHOR / 解像度: 10.0 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 3.1) 詳細: A composite 3D map of the crystal, generated by merging of hexagon-focused and six trimer-focused 3D maps (EMD-25737 and EMD-25738, respectively) in UCSF Chimera using the vop maximum command. 使用したサブトモグラム数: 7
抽出
トモグラム数: 62 / 使用した粒子像数: 105070
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1)
-
原子モデル構築 1
詳細
Model for fitting was generated by AlphaFold using the construct's amino acid sequence. Flexible fitting into corresponding densities was performed using ISOLDE tool in ChimeraX. The resulting structures were copied and fitted as rigid bodies into the 3D map by the "fit in map" function in Chimera.
精密化
プロトコル: FLEXIBLE FIT
得られたモデル
PDB-7t81: Model of Munc13-1 C1-C2B-MUN-C2C 2D crystal between lipid bilayers.