EMPIAR-10307 - Human pre-B spliceosome and U4/U6.U5 tri-snRNP [multiple data sets in MRC and MRCS formats]

EMPIAR-10307

Human pre-B spliceosome and U4/U6.U5 tri-snRNP [multiple data sets in MRC and MRCS formats]

Publication:

Mechanism of 5' splice site transfer for human spliceosome activation

Charenton C, Wilkinson ME, Nagai K

Science (New York, N.Y.) 364 (2019) 362-367

PMID: 30975767

DOI: 10.1126/science.aax3289

Related EMDB entry:

EMD-4687 EMD-4688 EMD-4675 EMD-4676 EMD-4686 EMD-4689 EMD-4690 EMD-4673 EMD-4674 EMD-4672 EMD-4658 EMD-4665

Deposited:

2019-04-12

Released:

2019-09-30

Last modified:

2019-09-30

Imageset size:

2.28 TB

Imageset DOI:

10.6019/EMPIAR-10307

Experimental metadata:

Download xml json

Contains:

micrographs - single frame
picked particles - single frame - processed
picked particles - single frame - unprocessed

Image Sets

1. Dataset 1 of human pre-B spliceosome; motion-corrected micrographs

Category:

micrographs - single frame

Image format:

MRC

No. of images or tilt series:

4627

Image size:

(3838, 3710)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.025, 1.025)

Details:

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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2. Dataset 2 of human pre-B spliceosome; motion-corrected micrographs

Category:

micrographs - single frame

Image format:

MRC

No. of images or tilt series:

6869

Image size:

(3838, 3710)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.022, 1.022)

Details:

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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3. Dataset 3 of human pre-B spliceosome; motion-corrected micrographs

Category:

micrographs - single frame

Image format:

MRC

No. of images or tilt series:

7303

Image size:

(3838, 3710)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.02, 1.02)

Details:

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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4. Dataset 4 of human pre-B spliceosome; motion-corrected micrographs

Category:

micrographs - single frame

Image format:

MRC

No. of images or tilt series:

6672

Image size:

(3838, 3710)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.022, 1.022)

Details:

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)

Files:

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5. Dataset 5 of human pre-B spliceosome; motion-corrected micrographs

Category:

micrographs - single frame

Image format:

MRC

No. of images or tilt series:

3234

Image size:

(3838, 3710)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.022, 1.022)

Details:

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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6. Selected U4/U6.U5 tri-snRNP particles after Bayesian polishing

Category:

picked particles - single frame - processed

Image format:

MRCS

No. of images or tilt series:

20537

Image size:

(420, 420)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.022, 1.022)

Details:

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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7. Crude shifted preB particles

Category:

picked particles - single frame - unprocessed

Image format:

MRCS

No. of images or tilt series:

873597

Image size:

(360, 360)

Pixel type:

32 BIT FLOAT

Pixel spacing:

(1.7033, 1.7033)

Details:

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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