REL 2024-10-03 2025-06-13 2025-06-13 0000-0003-2580-2299 Michael Grange Rosalind Franklin Institute Harwell campus Oxfordshire United Kingdom OX11 0QX 0000-0003-2580-2299 Michael Grange Rosalind Franklin Institute Harwell campus Oxfordshire United Kingdom OX11 0QX Berger C. Watson H. Naismith J. H. Dumoux M. Grange M. Wellcome Trust 220526/Z/20/Z United Kingdom Wellcome Trust 225902/Z/22/Z United Kingdom 2.2 10.6019/EMPIAR-12413 EMDB cell EMD-50675 EMD-50699 EMD-50700 EMD-50701 EMD-50702 EMD-50703 EMD-50676 EMD-50678 EMD-50679 EMD-50680 EMD-50681 EMD-50682 EMD-50683 EMD-50684 EMD-50685 EMD-50686 EMD-50687 EMD-50688 EMD-50689 EMD-50690 EMD-50691 EMD-50692 EMD-50693 EMD-50694 EMD-50695 EMD-50696 EMD-50697 EMD-50698 EMD-50704 EMD-50705 EMD-50706 Berger C Watson H Naismith J Dumoux M Grange M bioRxiv bioRxiv United States 2024 English 10.1101/2024.06.20.599830

286 tilt-series acquired on xenon plasma FIB lamellae with a Titan Krios G4 equipped with a Selectris energy filter and Falcon 4 camera recorded in EER format. Data was used to obtain a 4.0 Å structure of the E. coli ribosome.

Berger C Watson H Naismith JH Dumoux M Grange M Nature communications Nat Commun 1 16 2025 English 10.1038/s41467-025-57493-3 40055361 .eer format tilt-series /data/raw_data/eer micrographs - multiframe TIFF EER 286 10 UNSIGNED BYTE 16384 0.95 16384 0.95

1) data description 2) reconstruction and processing parameters 1) file structure data/ -->tomograms.xlsx #Excel file with statistics on tomograms and STA process -->tomograms.txt #text file with statistics on tomograms and STA process -->data_readme.txt #information on uploaded files, reconstruction and processing parameters raw_data/ eer/ -->fractions.eer #fractions in .eer format, recorded with tomo5, and renamed using a script. Calibrated Pixel size for 4K= 1.9A. mdocs/ -->excludedtilts.txt #list of tilts excluded during data processing original/ -->TIR???.mdoc #mdocs files saved by tomo5, and renamed using a script tilts_excluded/ -->TIR???.mdoc #selection of mdocs for all tilt-series retained for STA, with tilts listed in excludedtilts.txt removed gain_reference/ -->20230405_092712_EER_GainReference.gain #original gain reference recorded for dataset -->20230405_092712_EER_GainReference.mrc #gain reference converted to .mrc using eman2, for use in Warp/M processed_data/ search_maps/ #search maps, stitched search maps, segmentations for amorphous layers reconstructed_tomograms/ AlignZvalues.txt #list of AlignZ values used for reconstruction for each tomogram SART/ #tomograms reconstructed by AreTomo (SART) isonet/ #SART reconstructed tomograms, filtered with Isonet model trained on data subset alignment_files/ TIR???/TIR???.xf #.xf alignment files generated by Aretomo TIR???/TIR???.st #.st tilt-series generated by Warp (removed tilts excluded) TIR???/TIR???.rawtlt #.rawtlt files generated by warp (removed tilts excluded) surface_models/ TIR???.mod #imod model files for lamella surface boundaries (manually picked every ~100 slices + interpolated) TIR???.txt #txt file format of boundary model (converted with imod model2point) isonet_model/ -->isonet_model_v1.h5 #isonet model trained on tomograms from this dataset, and used for isonet filtering cryolo/ training_annotations/ #manually picked coordinates, used for training cryolo models -->cryolo_model_unfiltered01.h5 #cryolo ribosome model for picking on SART reconstructions -->cryolo_model_filt01.h5 #cryolo ribosome model for picking on bandpass filtered reconstructions -->cryolo_model_deconv01.h5 #cryolo ribosome model for picking on EM deconvolved reconstructions -->cryolo_model_isonet01.h5 #cryolo ribosome model for picking on isonet filtered reconstructions star_files/ -->20231016_xenon_ribo_iso.star #star file after cryolo prediction on isonet filtered tomograms -->20231219_perparticle_distance.star #star file post-M, with additional columns for e.g. distance to PFIB surfaces, backside lamella Aretomo reconstruction parameters (Aretomo version 1.2.5): -inMrc TIR???.st \ #generated by warp -OutMrc TIR???.mrc \ -AngFile TIR???.rawtlt \ #generated by Warp -VolZ 3096 \ -OutBin 8 \ -PixSize 1.9 \ -Gpu 0 \ -DarkTol 0.000001 \ #effectivly disable exclusion of tilts by Aretomo -AlignZ "$thickness" \ #value from AlignZvalues.txt were used for each tomogram -TiltCor 0 20 \ -OutXF 1 \ -->Important note on handedness<-- To obtain the correct handedness in Warp for both the map and during CTF processing, we did the following steps. 1)after .eer processing in Warp, run the .tomostar step. During the first "import tilt-series from IMOD" step where .st and .rawtlt files are exported, make sure that the "Don't invert tilt angles" box is NOT ticked. 2)After Aretomo reconstruction, tomograms are flipped using the imod command trimvol -yz, to match the handedness of tomograms reconstructed by Warp 3)After tomogram reconstruction generating the .xf alignment files, run the "import tilt-series from IMOD" step again, make sure to TICK the "Don't invert tilt angles" box 4)The map handedness is now correct, but the handedness is incorrect for CTF estimation. Start processing a single tomogram and click stop processing. Go to the Fourier Space tab and run the check tilt handedness feature. This will give a negative correlation and recommend flipping the handedness. Click OK to flip the handedness for CTF estimation. This will not affect the map handedness. Slightly adjust the CTF fitting range (to reset the tilt-series that was already processed) and rerun processing.