Volume 110, Issue 4, Pages 850-859 (February 2016) Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms  Jose-Jesus Fernandez, Ulrike Laugks, Miroslava Schaffer, Felix J.B. Bäuerlein, Maryam Khoshouei, Wolfgang Baumeister, Vladan Lucic  Biophysical Journal  Volume 110, Issue 4, Pages 850-859 (February 2016) DOI: 10.1016/j.bpj.2015.10.043 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Surface contamination and artifact removal. (a–c) Untilted image of the initial tilt-series, z slice, and y slice of the tomogram, respectively, showing surface contamination and the artifacts induced in the reconstruction. (d–f) Restored tomogram obtained by the artifact-removing procedure. The contamination was removed from the tilt-series, which caused the ripples to disappear from the tomogram. (Solid arrows) Pointers to contamination in the tilt-series (a) and tomogram (c). (Open arrows) Pointers to the corresponding positions in the restored versions (d and f). (Small lines on the left, b and c, e and f) Correspondence between the z- and y slices of the tomograms. Scale bar = 100 nm. Biophysical Journal 2016 110, 850-859DOI: (10.1016/j.bpj.2015.10.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Evaluation of the artifact-removing procedure. (a) Standard deviation of the contamination region of the tomogram at different iterations. Most of the contamination is removed at the first 3–5 iterations. (b) Tests with a phantom composed of vertical lines with defined spatial frequency. Phantom distorted by artifacts (left) and the restored tomogram (right) were obtained in two different ways: the phantom was superposed in the whole (top) and on the contamination-only tomogram (bottom). The artifact-removing procedure recovered the phantom in both cases. (c) Subtomogram averaging test. (On the right) Gallery of ribosomes (z- and y slices) in (top) control, a tomogram of ribosomes distorted by contamination (middle), and after the application of the procedure (bottom). (On the left) Restored versus control (solid) and distorted versus control (shaded) FSC curves and the subtomogram averages (insets show two orthogonal planes of each volume) from restored (top), distorted (bottom), and control (middle, right) tomograms. Scale bars = 100 nm. Biophysical Journal 2016 110, 850-859DOI: (10.1016/j.bpj.2015.10.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Applications to samples affected by Pt-sputtering and large contamination. (a–c) A tomogram of HeLa cells containing Pt-sputtering-induced artifacts. (a) Tomogram before (left) and after the artifact-removing procedure (right). The z- and y slices are shown (top, bottom, respectively). (Dashed square) Area zoomed in (b) (arrow pointing to ripples). (c) Zoomed areas of another tomogram obtained under the same conditions, before and after the application of procedure. (d) A tomogram of neuronal synapse containing two medium/large contamination granules (denoted by arrowheads). Initial (left) and restored (right) tomograms are shown. (Top-left, bottom-left, and top-right panels) The z-, y-, and x slices, respectively. (Solid arrows) Pointers to ripples caused by the medium/large contamination granules. (Open arrows and arrowheads) Pointers to the corresponding positions in the restored tomograms. (Insets, with arrows) Zoomed views of the highlighted areas. (Small lines on the left, a and d) Correspondence between the x-, y-, and z slices of the tomograms. Scale bars = 100 nm. Biophysical Journal 2016 110, 850-859DOI: (10.1016/j.bpj.2015.10.043) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Applications to samples that exhibit a common, weak granular surface contamination. The untilted image (a) and the tomogram (b) of neuronal cultures show an almost continuous granular layer. The artifacts in the y slice of the tomogram in (b) arise from the interference between the streaks of the fine surface contamination and those of gold particles. (Solid arrows) Pointers to streak artifacts. Images were acquired with a CCD camera. (c–e) Sparse contamination, images acquired with a DDD camera. (c) Untilted image of the tilt-series. (d and e, arrows) Pointers to the two y slices of the tomogram where streak artifacts originated by the contamination granules. In all cases, the procedure removed or attenuated the artifacts (initial tomograms are on the top and the restored on the bottom in b, d, and e). (Open arrows) Pointers to positions on the restored tomograms that correspond to the contamination. (Insets) Differences between the distorted and restored details (outlined by dashed lines). Scale bars = 100 nm. Biophysical Journal 2016 110, 850-859DOI: (10.1016/j.bpj.2015.10.043) Copyright © 2016 Biophysical Society Terms and Conditions