micrographs - multiframe

EER

286

(16384, 16384)

UNSIGNED BYTE

(0.95, 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.

Download #files