3D reconstruction of protein kinase using 2DTM-derived particle stack.

(a) From left to right: the single-particle reconstruction (EMDB:0409), the 2DTM template, and the 2DTM-derived reconstruction of the protein kinase (29). The x-ray structural model of the protein kinase (PDBID: 1ATP) is also shown where ATP, Mn2+, water molecules, and a segment of alpha-helix (residues 222-227) were deleted from the model. (b) Angular distribution plot and FSC curve calculated using cisTEM. (c) Densities at the ATP-binding site and deleted residues 222–227 visualized by ChimeraX (57). The average Q-scores between the 2DTM reconstruction and the X-ray model was calculated using the MapQ command line tool (33, 34).

2DTM-based reconstruction of the ATP-binding pocket.

Each row shows, from left to right: (1) the atomic model used to generating the template, with residues within a specified radius from ATP deleted (highlighted in blue); (2) the 2DTM reconstruction, fitted with the template model and colored by the nearest atoms; (3) the recovered ATP density and its average Q-score in the 2DTM reconstruction; and (4) the average Q-scores of all residues.Residues omitted from the templates are circled and colored by chain identity. Q-scores were calculated using the MapQ command line tool (33, 34). (a) Residues within 3 Å of ATP were deleted. (b) IP20 and residues within 3 Å of both ATP were deleted. (c) Residues within 5.5 Å of ATP were deleted.

RELION processing of 2DTM-derived particle stack.

(a) RELION skip-alignment 3D classification of the 2DTM-derived particle stack in Fig. 1. Classes 1-4 were merged for 3D refinement. (b) Both skip-alignment and alignment-enabled auto-refinement were performed on the selected particles from (a). In both maps, densities for the ATP and backbone of the deleted residues were recovered.

Image statistics of the untilted micrographs in EMPIAR-10252.

(a) CTF fitting scores for 2,488 untilted images, calculated using ctffind5. Images with scores above 0.2 or below 0.05 were excluded from 2DTM searches. (b) Mean defocus values of the 2,314 images retained for 2DTM. (c) Sample thickness estimates from ctffind5, with a median thickness of 363 Å. (d) Particle counts per thickness bin, based on 17,274 particles extracted from the 2,314 images using extract-particles. Particle counts per thickness bin after particle selection using filter-particles, showing the final stack of 7,353 particles.

Examples of micrographs excluded from 2DTM search.

(a) Very low contrast and ice contamination. (b) Extremely low contrast, likely drift or astigmatism. (c) Particle aggregation or contamination. (d) Crystalline ice or fractured film.

2DTM reconstructions with varying particle selection parameters.

From left to right, particle stacks were generated using the following thresholds: (a) 2DTM search template where ATP, Mn2+ and residues 222-227 (blue) were deleted; (b) 2DTM p-value = 8.0, (c) 2DTM p-value = 7.0; (d) 2DTM SNR = 7.5; (e) 2DTM p-value = 8.0 with tilted data. All other parameters were kept constant as in Fig. 1. For the tilted dataset, only images with CTF scores between 0.04 and 0.13 and estimated thickness between 100 and 1000 Å were selected. The number of particles in the final stack and the Q-scores of the recovered ATP densities are indicated in the figures. Template bias were calculated using a in-house Python script adapted from (28).

2DTM reconstruction using the AlphaFold model as the search template.

(a) Left: Structural comparison between the X-ray model (PDB ID: 1ATP, gray) and the AlphaFold3 predicted model (blue) (38). Residues within 3 Å of ATP were deleted from the template and now shown. Right: 2DTM-derived maps using the AlphaFold3 template (blue) and the X-ray template (yellow). (b) Reconstruction at the ATP-binding site using the X-ray model (top) and the AlphaFold3 model (bottom) as the template (contour level σ = 5). (c) Reconstruction at residue 18 (ARG) on IP20 using the X-ray model (top) and the AlphaFold3 model (bottom) as the template (contour level σ = 4).

Single-particle cryo-EM lower molecular weight limits under different assumptions.

A constrained search restricts the x and y dimensions to a 5-by-5 pixel window.

Theoretical lower molecular weight limit.

A constrained search restricts the x and y dimensions to a 5-by-5 pixel window. At the minimal molecular weight, the SNR calculated from alignment noise and phase contrast are equal.

Difference map between the template and the reconstruction shown in Fig. 1.

(a) The difference map (pink) was generated using the diffmap program (58). Contour levels are set to 5 (left) and 8 (right). (b-c) At a contour level of 8, difference densities are observed at the ATP binding site and residues 222-227.