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- EMDB-18727: Cryo-EM structure of the cross-exon pre-B+AMPPNP complex (tri-snR... -

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Basic information

Entry
Database: EMDB / ID: EMD-18727
TitleCryo-EM structure of the cross-exon pre-B+AMPPNP complex (tri-snRNP region)
Map data
Sample
  • Complex: human spliceosomal pre-B+AMPPNP complex
Keywordsspliceosome / SPLICING
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsZhang Z / Kumar V / Dybkov O / Will CL / Zhong J / Ludwig S / Urlaub H / Kastner B / Stark H / Luehrmann R
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Nature / Year: 2024
Title: Structural insights into the cross-exon to cross-intron spliceosome switch.
Authors: Zhenwei Zhang / Vinay Kumar / Olexandr Dybkov / Cindy L Will / Jiayun Zhong / Sebastian E J Ludwig / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann /
Abstract: Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron. Alternatively, it can occur ...Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron. Alternatively, it can occur through an exon-defined pathway, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex. Exon definition promotes the splicing of upstream introns and plays a key part in alternative splicing regulation. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.
History
DepositionOct 24, 2023-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18727.png

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Map

FileDownload / File: emd_18727.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 480 pix.
= 648. Å		1.35 Å/pix.
x 480 pix.
= 648. Å		1.35 Å/pix.
x 480 pix.
= 648. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.02448405 - 0.07110691
Average (Standard dev.)0.00013808579 (±0.0029200322)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 648.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_18727_half_map_1.map
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AxesZYX

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Half map: #2

Fileemd_18727_half_map_2.map
Projections & Slices
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Sample components

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Entire : human spliceosomal pre-B+AMPPNP complex

EntireName: human spliceosomal pre-B+AMPPNP complex
Components
  • Complex: human spliceosomal pre-B+AMPPNP complex

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Supramolecule #1: human spliceosomal pre-B+AMPPNP complex

SupramoleculeName: human spliceosomal pre-B+AMPPNP complex / type: complex / ID: 1 / Parent: 0
Macromolecule list: #1-#8, #11-#12, #14, #9-#10, #13, #15-#34
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53422
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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