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

(a) From left to right: the single-particle reconstruction (EMDB:0409) (30), the 2DTM template, and the 2DTM-derived reconstruction of the protein kinase. (b) Angular distribution plot and FSC curve calculated using cisTEM. Note that these FSC curves are not gold-standard FSCs, as the reconstruction uses orientations determined by 2D template matching rather than independent half-set refinement. FSCuncor denotes the uncorrected FSC computed within a generous mask, which crosses the 0.143 threshold at 3.0 Å, and “Particle FSC” denotes the solvent-corrected FSC obtained using the mask-volume correction factor as described in the cisTEM/Frealign framework (63). (c) Densities at the ATP-binding site and deleted residues 222–227. The X-ray structural model (PDBID: 1ATP) (31) is shown with atoms retained in the template colored grey and deleted atoms colored blue. The alpha-helical model for residues 222–227 is rendered transparent to better show the recovery of the helical turn in the reconstruction. The average Q-scores between the 2DTM reconstruction and the X-ray model were calculated using the MapQ command line tool (34, 35). Q-scores for ATP, Mn2+, and deleted residues 222–227 were 0.60, 0.48/0.73, 0.63, 0.51, 0.57, 0.54, 0.61, and 0.53, respectively.

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

Each row shows, from left to right: (1) the atomic model used to generate the template, with residues within a specified radius from ATP deleted (highlighted in blue); (2) the 2DTM reconstruction fitted with the full model, with labeled residues corresponding to those circled in red in the Q-score plot; (3) the recovered ATP density fitted with the full model; and (4) backbone Q-scores of all residues. Omitted residues are shown in blue (circles for chain E, diamonds for IP20); kept residues in gray. ATP and Mn2+ are shown in orange. Red circles indicate the omitted residues with the lowest backbone Q-scores, corresponding to the labeled residues in column 2. Q-scores were calculated using the MapQ command line tool (34, 35). (a) Residues within 3 Å of ATP were deleted. (b) IP20 and residues within 3 Å of ATP were deleted. (c) Residues within 5.5 Å of ATP were deleted. Map contour σ = 5.

RELION processing of 2DTM-derived particle stack.

(a) RELION 3D classification without angular refinement of the 2DTM-derived particle stack from Figure 1, using five classes (Tau_fudge=4, angular limit 7 Å). Classes are colored individually and labeled with particle count, percentage of the dataset, and estimated resolution. Classes 1–4 were merged for 3D reconstruction; Class 5 (156 particles, ∼2%, ∼20.5 Å) was excluded. Map contour σ = 5. (b) Comparison of 3D reconstructions using 2DTM orientations directly (top row, green) and RELION auto-refine (bottom row, red). From left to right: full map, zoomed view of the ATP binding pocket and deleted residues 222–227 with the atomic model (blue) overlaid (map contour σ = 7), orientation distribution, and Fourier shell correlation (FSC) curves. The 2DTM-orientation reconstruction reaches 3.1 Å and the RELION auto-refined reconstruction 3.7 Å at the FSC=0.143 threshold. In both maps, densities for ATP and the backbone of the deleted residues were recovered, but the 2DTM-derived orientations yielded sharper and more continuous density in the omitted regions. (c) RELION auto-refinement resolution as a function of the initial low-pass filter (--ini_high). The final row includes all five classes (i.e., keeping Class 5), yielding 3.7 Å, identical to the reconstruction without Class 5.

2DTM reconstructions with varying particle selection parameters.

The first column shows the template model with the X-ray structure overlaid (deleted atoms in blue). The remaining columns show reconstructions using different selection thresholds (map contour σ = 5). The top and bottom rows show zoomed views of the ATP-binding pocket and deleted residues 222–227, respectively. Each column header lists the number of particles and the template bias Ω, defined as Ω = (∑mask Vfull − ∑mask Vomit)/ ∑mask Vfull, where Vfull and Vomit are reconstructions using orientations and particles derived from independent 2DTM searches with the full and omit templates, respectively, and the sum is restricted to the omission mask derived from the difference between the two templates. Ω = 0 means the omit reconstruction recovers the same density (no bias from the template), while Ω = 1 means all density disappears without the template. Q-scores for ATP and residues 222–227 are reported in Figure 4—source data 1. Template bias was calculated using the 2DTM_postprocess_tool Python package, adapted from (29). The omission mask used to compute Ω is shown in Figure 4—figure supplement 1.

Site-specific composite omit map assembled from 36 partial-deletion reconstructions.

Each of the 36 omit templates deletes ∼10 non-overlapping residues. For each template, local density was extracted within 3 Å of the omitted atoms, with neighboring residues (i±1) excluded using a 2 Å cutoff on backbone Cα atoms. Local density patches were assigned uniquely and merged such that each voxel is contributed only by the reconstruction in which the corresponding region was omitted from the template. (a) Atomic model of the protein kinase (PDBID: 1ATP). (b) Left: composite omit map. Right: zoomed view of the ATP-binding pocket. (c) Left: composite omit map low-pass filtered to 4 Å. Right: zoomed view of the ATP-binding pocket. The composite demonstrates that density can be recovered at distributed locations across the protein, including peripheral and surface-exposed regions, although the quality of recovery varies across sites. Map contour σ = 4.

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

(a) Structural comparison between the X-ray model (PDB ID: 1ATP, gray) and the AlphaFold3 predicted model (tan) (39). Residues within 3 Å of ATP were deleted from both templates and not shown. RMSD between the undeleted structures is 0.45 Å. (b) 2DTM-derived maps using the AlphaFold3 template (tan) and the X-ray template (gray). (c) Reconstruction at the ATP-binding site using the X-ray model (top) and the AlphaFold3 model (bottom) as the template (map contour σ = 5). (c) Reconstruction at residue 18 (ARG) on IP20 using the X-ray model (top) and the AlphaFold3 model (bottom) as the template (map contour 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.

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 367 Å. Negative thickness estimates were excluded from the histogram. (d) Particle counts per thickness bin, based on 17,274 particles extracted from the 2,314 images using extract-particles. (e) 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) Cross-grating calibration grid.

Effect of template resolution on reconstruction quality.

Comparison of 3D reconstructions using particles aligned at bin2x (1.117 Å/pixel) versus bin4x (2.234 Å/pixel) resolution. For the bin4x experiment, 2DTM was performed on bin4x images and the detected particle coordinates were used to extract particles from the bin2x images for reconstruction. Densities at the ATP-binding site and deleted residues 222–227 are shown. The bin4x-aligned reconstruction shows loss of ATP density and degraded backbone density for residues 222–227, demonstrating that high-resolution signal in the template is critical for accurate particle alignment and recovery of omitted densities. Map contour σ = 5.

Difference map between the template (gray, map contour σ = 15) and the reconstruction shown in Figure 1.

The difference map was generated using the diffmap.exe program (53) with a protein soft mask. Positive (pink) and negative (light blue) difference densities are shown at map contour σ = 20. Positive densities indicate regions present in the reconstruction but absent from the template, with coherent features at the ATP binding site and residues 222–227, which were deleted from the template. Residual noisy densities elsewhere reflect limitations of the forward model.

Grouped occupancy refinement of omitted and control residues.

Occupancies were refined using Phenix real-space refinement against the omit reconstruction (Figure 1), with the full 1ATP model docked into the map. Omitted residues (222–227), ATP, and Mn2+ were absent from the template used for 2DTM alignment. Control residues (150–155) were included in the template. Occupancies near 1.0 for control residues and intermediate values (0.55–0.80) for omitted residues confirm partial, unbiased recovery of the omitted-region density.

FSC comparison between cisTEM and RELION on the same half-maps.

Both curves were computed from the same cisTEM half-maps (Figure 1 reconstruction). The cisTEM Particle FSC (blue) uses a spherical mask with an analytical solvent-fraction correction, while the RELION masked FSC (orange) uses a tight 3D protein mask applied directly to the half-maps. Both cross the FSC = 0.143 threshold at ∼2.6–2.7 Å, confirming that the two packages give consistent resolution estimates when applied to the same data.

Q-scores for omitted regions across particle selection conditions (Figure 4).

Values are average Q-scores over all atoms in each group, calculated using the MapQ command line tool (34, 35). The ATP Q-score is averaged over all 31 non-hydrogen atoms; the residues 222–227 Q-score is averaged over all non-hydrogen atoms in the six residues (Trp, Ala, Leu, Gly, Val, Leu).

Central section of the omission mask used for template bias (Ω) calculation.

The mask was derived from the difference between the full and omit templates (threshold = average of top-100 voxels / 5; 959 masked voxels). Left: full template. Center: omit template (residues 222–227, ATP, Mn, and H2O deleted). Right: binary omission mask, with the two omitted regions (ATP + Mn and residues 222–227) annotated. The mask isolates the omitted region for the Ω calculation shown in Figure 4. Voxel size 1.117 Å.

Map–model FSC for X-ray and AlphaFold3 templates in Figure 6.

Map–model FSC was computed between each atomic model-derived template and the corresponding 2DTM reconstruction using cisTEM calculate_fsc, with a protein envelope mask. Left: X-ray-derived template versus X-ray-derived 2DTM reconstruction. Right: AlphaFold3-derived template versus AlphaFold3-derived 2DTM reconstruction. Both curves cross the FSC = 0.143 threshold at ∼2.3 Å.