Cryo-EM data collection

Protocol for sample preparation, Cryo-EM data collection and particle picking

Preparation of Sample

The complex of NatB (NAA25 and NAA20) was obtained by co-expression of both genes in sf9 cells (see method section), and complex formation was confirmed by gel filtration and SDS- PAGE (see Figure 1A). Complex formation was further validated by confirming N-terminal acetyltransferase activity of the purified NatB. Bisubstrate inhibitor binding to the complex was confirmed by IC-50 assay (Figure 1C). Excess Bisubstrate analogue was added into the NatB complex right before the cryo-grids were made (the EM volume of the bisubstrate analogue is shown in Figure 4B). Together, this confirmed that the complex of NAA20, NAA25 and bisubstrate analogue was formed, in agreement with the overall EM map and PDB model (see supplementary Figure 2).

Data Collection

Data collection was performed as described in the methods section - “Titan Krios equipped with a K3 Summit direct detector (Gatan), at a magnification of 105,000×, with defocus values from −0.1 to −2.0 µm. Each stack was exposed in super-resolution mode with a total dose of 45 e-/Å2, resulting in 35 frames per stack. Image stacks were automatically collected with Latitude software (Gatan, Inc)”

Data collection parameters are indicated below.

 

Picking of particles

Particle picking was carried in with Relion 3.0, as described “About 3000 particles were manually picked to generate several rough 2D class averages. Representative 2D classes were used to automatically pick ~1,927,673 particles from 5281 micrographs in Relion 3.0”. The reader may also use the 3D EM map that we have deposited in EMPIAR (ID: 10477) as a template to do particle autopicking in Relion 3.0.