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Tilt pair images were recorded by turning the sample holder around the tilt axis by 0° and 10°.
(A ) Representative fields from a random conical tilt pair of particles assembled with Rev-RRE, Crm1, and RanGTP.
By assessing the consistency between the orientations from independently refined tilt pair particles with the known microscope tilt geometry, we estimated an alignment precision for the tilt pairs of 2° (Rosenthal and Henderson, 2003).
(F ) Same for the DNA-bound complex (G ) Tilt pair validation using 267 particle pairs that were selected from 20 image pairs collected at 0 and 20° tilt angle of the sample stage.
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For RCT data sets, particle tilt pairs were identified using TiltPicker (Voss et al., 2009).
We recorded tilt pairs of videos on the 70S T. thermophilus ribosome sample and used 2D and 3D classification to select a structurally homogeneous subpopulation of 15,202 particle pairs for 3D reconstruction (see 'Materials and methods').
Because the alignment accuracy of individual particles remains unknown in typical single-particle analysis, we collected an entire data set as tilted pairs and used tilt-pair analysis to experimentally assess alignment errors for video frames (Rosenthal and Henderson, 2003).
For each recline angle, center of pressure displacements was significantly different between two of the three tilt angle pairs.
To help solve these problems, we have developed an automated system for the rapid acquisition of accurately aligned and focused tilt pairs.
We prepared cryo-negatively stained samples on Affinity Grids and collected 50°/0° image tilt pairs (Figure S3A).
Tilt pairs were collected at tilt angles of 0° and 10°.
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