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![[Figure 4]](rr5256fig4mag.jpg)
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Figure 4
Constrained searches improve the sensitivity and functional resolution of 2DTM. Constraining the search space in 2DTM by limiting the number of cross-correlograms through known molecular positions and orientations decreases the signal-to-noise ratio (SNR) threshold for detection, thereby enabling the identification of smaller molecular complexes. (a) Plot showing the minimum detectable molecular mass at a false-positive rate of 1 in 200 particles, relative to the standard threshold of one false positive per micrograph. When both position and orientation are known, the detection threshold improves by approximately tenfold. (b) Plot showing the number of detections in 70 micrographs when performing an unconstrained search for the LSU and SSU body or constraining the search for the SSU body using positions and orientations from the LSU search (right). (c) Simulated slab of LSU particles (blue) and SSU particles (orange) found using the unconstrained 2DTM search overlaid on a representative micrograph. (d) The same LSU particles and micrograph now depicted with the constrained search SSU particles (green) showing that the constrained search increases 2DTM sensitivity. (e) Reconstructed volume of the LSU particles where a SSU particle had also been detected. The SSU is clearly present in the reconstruction despite not being present in the template. The PDB model 6q8y was docked into the map and a region of the SSU body (18S rRNA helix 44) is highlighted to demonstrate the fit between the map and the model. To estimate the resolution of the SSU body, a mask was created around the SSU body (green). This region was reconstructed to 5.1 Å resolution, as determined by a Fourier shell correlation of 0.143 (f) (Rosenthal & Henderson, 2003  ). |
![[Figure 4]](rr5256fig4.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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