Extended Data Fig. 1: CryoEM data processing workflow for the FeSII-nitrogenase complex.
From: Structural basis for the conformational protection of nitrogenase from O2
Single-particle cryoEM data processing workflow for (left to right) 2:2:1 complex (2.27 Å, C2 symmetry), as-isolated MoFeP (2.67 Å, C2 symmetry), 1:1:1 complex (3.94 Å, C1), and terminal end-piece of 2:2:1 complex (2.83 Å, C1) obtained from the same O2-exposed sample prepared with 10 μM MoFeP, 20 μM FeP, 20 μM FeSII, 5 mM DT, and 5 mM MgATP. Briefly, particle coordinates were obtained via template picking either with MoFeP or ternary complex. Picked particles were subject to 2-D classification before re-extraction based on the desired box size of the targeted structure. Ab initio model generation as well as 3-D classification were used to sort heterogeneity in-between multiple rounds of NU-refinement. The 2:2:1 complex (far left) also utilized RBMC before final refinement. The terminal 2:2:1 complex (far right) utilized local masking and refinement to sort only terminal complex particles. Each of the final structures is colored by local resolution and the 3-D FSC plots are shown.
