Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 57 electrons per squared Angstrom over 8 seconds. Data collected on Titan Krios 2 at the Astbury Biostructure Laboratory, University of Leeds, on a K2 detector operated in counting mode. dataset1_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)
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Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 50 electrons per squared Angstrom over 6 seconds. Data collected on Titan Krios 3 at the MRC Laboratory of Molecular Biology on a K2 detector operated in counting mode. dataset2_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)
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Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 55.4 electrons per squared Angstrom over 12 seconds. Data collected on a Titan Krios at the University of Cambridge biochemistry department on a K2 detector operated in counting mode. dataset3_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)
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Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 48.6 electrons per squared Angstrom over 6 seconds. Data collected on Titan Krios 3 at the MRC Laboratory of Molecular Biology on a K2 detector operated in counting mode. dataset4_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)
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Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 44 electrons per squared Angstrom over 6 seconds. Data collected on Titan Krios 3 at the MRC Laboratory of Molecular Biology on a K2 detector operated in counting mode. dataset5_gautomatchpicking.star contains particle coordinates picked by Gautomatch using templates from pre-B complex 2D averages from the other 4 datasets (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)
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Particles from datasets 2, 3, and 4 were picked with CRYOLO (see dataset*_CRYOLOpicking.star) and 3D classified. Tri-snRNP and pre-B particles were selected, CTF-refined and subjected to Bayesian polishing in RELION 3.0, giving the particle stacks in Polish/ A further round of CTF refinement produced the reconstruction deposited in the EMDB as EMD-4658, with the resultant run_data.star file deposited here as "trisnRNP_EMD4658_refine_data.star"
Further classification of these particles yielded various other deposited maps, metadata for which are deposited here. In summary:
"preB_unshiftedbox_EMD4665_refine_data.star" The subset of these polished tri-snRNP particles that correspond to pre-B complex, corresponds to EMD-4665
"trisnRNP_prp28class_EMD4686_refine_data.star" Subset of tri-snRNP with strong Prp28, corresponds to EMD-4686
"trisnRNP_U540Kclass_refine_data.star" Subset of tri-snRNP with strong U5-40K
For full details see Charenton et al Science 2019.
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Particles from all datasets were picked (see dataset*_*picking.star) and 3D classified using a tri-snRNP reference. Tri-snRNP and pre-B particles were selected and re-extracted with a "shifted box" such that the box would encompass the full pre-B complex. Particles were also scaled down to 1.7033 Ångstrom per pixel. Further rounds of 3D classification produced a crude set of 117,284 pre-B particles, metadata in the deposited preB_allparticles_shiftedbox_refine_data.star Further classification of these particles yielded various deposited maps, metadata for which are deposited here. In summary:
"preB_prp4kclass_EMD4688_refine_data.star" The subset of pre-B particles with strong density for Prp4 kinase, corresponds to EMD-4688
"preB_U1class_EMD4687_refine_data.star" The subset of pre-B particles with strong density for U1 snRNP, corresponds to EMD-4687
"preB_U2class_EMD4690_refine_data.star" The subset of pre-B particles with strong density for U2 snRNP, corresponds to EMD4690
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