Journal: Nature / Year: 2017 Title: Structure of a pre-catalytic spliceosome. Authors: Clemens Plaschka / Pei-Chun Lin / Kiyoshi Nagai / Abstract: Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy ...Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.
2: U2 snRNA 3: U6 snRNA-associated Sm-like protein LSm3 4: U4 snRNA 5: U5 snRNA 6: U6 snRNA 7: U6 snRNA-associated Sm-like protein LSm7 8: U6 snRNA-associated Sm-like protein LSm8 A: Pre-mRNA-splicing factor 8 B: Pre-mRNA-splicing helicase BRR2 C: Pre-mRNA-splicing factor SNU114 D: Spliceosomal protein DIB1 E: 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component F: Pre-mRNA-processing factor 31 G: U4/U6 small nuclear ribonucleoprotein PRP3 H: U4/U6 small nuclear ribonucleoprotein PRP4 I: Yeast UBC4 gene for ubiquitin-conjugating enzyme J: Pre-mRNA-splicing factor 6 K: 13 kDa ribonucleoprotein-associated protein L: 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component M: Pre-mRNA-splicing factor 38 N: Pre-mRNA-splicing factor SPP381 O: U2 snRNP component HSH155 P: Pre-mRNA-splicing factor RSE1 Q: Cold sensitive U2 snRNA suppressor 1 R: Protein HSH49 S: Pre-mRNA-splicing factor RDS3 T: Pre-mRNA-splicing factor PRP9 U: Pre-mRNA-splicing factor PRP11 V: Pre-mRNA-splicing factor PRP21 W: U2 small nuclear ribonucleoprotein A' X: Unknown Y: U2 small nuclear ribonucleoprotein B'' Z: RDS3 complex subunit 10 a: U6 snRNA-associated Sm-like protein LSm2 b: Small nuclear ribonucleoprotein-associated protein B d: Small nuclear ribonucleoprotein Sm D3 e: Small nuclear ribonucleoprotein E f: Small nuclear ribonucleoprotein F g: Small nuclear ribonucleoprotein G h: Small nuclear ribonucleoprotein Sm D1 i: Small nuclear ribonucleoprotein Sm D2 j: U6 snRNA-associated Sm-like protein LSm4 k: Small nuclear ribonucleoprotein-associated protein B l: Small nuclear ribonucleoprotein Sm D1 m: Small nuclear ribonucleoprotein Sm D2 n: Small nuclear ribonucleoprotein Sm D3 o: U6 snRNA-associated Sm-like protein LSm5 p: Small nuclear ribonucleoprotein E q: Small nuclear ribonucleoprotein F r: Small nuclear ribonucleoprotein G s: Small nuclear ribonucleoprotein-associated protein B t: Small nuclear ribonucleoprotein Sm D1 u: Small nuclear ribonucleoprotein Sm D2 v: Small nuclear ribonucleoprotein Sm D3 w: Small nuclear ribonucleoprotein E x: Small nuclear ribonucleoprotein F y: Small nuclear ribonucleoprotein G z: U6 snRNA-associated Sm-like protein LSm6 hetero molecules
Mass: 22717.799 Da / Num. of mol.: 1 / Source method: isolated from a natural source Details: 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q12368
#22: Protein
U2snRNPcomponentHSH155
Mass: 110166.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U2 snRNP component HSH155 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P49955
#24: Protein
ColdsensitiveU2snRNAsuppressor1
Mass: 50210.699 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Cold sensitive U2 snRNA suppressor 1 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q02554
#25: Protein
ProteinHSH49
Mass: 24534.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Protein HSH49 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q99181
#31: Protein
Unknown
Mass: 7907.637 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Unknown protein helices within B complex / Source: (natural) Saccharomyces cerevisiae (baker's yeast)
#33: Protein
RDS3complexsubunit10 / Splicing factor 3b subunit
Mass: 10045.401 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RDS3 complex subunit 10 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P0C074
#35: Protein
Smallnuclearribonucleoprotein-associatedproteinB / snRNP-B / Sm protein B / SmB
Mass: 22426.990 Da / Num. of mol.: 3 / Source method: isolated from a natural source Details: Small nuclear ribonucleoprotein-associated protein B Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: P40018
Mass: 55974.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U4/U6 small nuclear ribonucleoprotein PRP3 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q03338
#15: Protein
U4/U6smallnuclearribonucleoproteinPRP4 / Pre-mRNA-processing protein 4
Mass: 52506.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U4/U6 small nuclear ribonucleoprotein PRP4 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P20053
+
U2 small nuclear ribonucleoprotein ... , 2 types, 2 molecules WY
#30: Protein
U2smallnuclearribonucleoproteinA' / U2 snRNP A' / Looks exceptionally like U2A protein 1
Mass: 27232.252 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U2 small nuclear ribonucleoprotein A' / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q08963
#32: Protein
U2smallnuclearribonucleoproteinB'' / U2 snRNP B''
Mass: 12850.944 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U2 small nuclear ribonucleoprotein B'' / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P40567
+
Small nuclear ribonucleoprotein ... , 6 types, 18 molecules dnvepwfqxgryhltimu
#36: Protein
SmallnuclearribonucleoproteinSmD3 / Sm-D3 / snRNP core protein D3
Mass: 11240.139 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein Sm D3 Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: P43321
#37: Protein
SmallnuclearribonucleoproteinE / snRNP-E / Sm protein E / SmE
Mass: 10385.098 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein E Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: Q12330
#38: Protein
SmallnuclearribonucleoproteinF / snRNP-F / Sm protein F / SmF
Mass: 9669.945 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein F Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: P54999
#39: Protein
SmallnuclearribonucleoproteinG / snRNP-G / Sm protein G / SmG
Mass: 8490.809 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein G Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: P40204
#40: Protein
SmallnuclearribonucleoproteinSmD1 / Sm-D1 / snRNP core protein D1
Mass: 16296.798 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein Sm D1 Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: Q02260
#41: Protein
SmallnuclearribonucleoproteinSmD2 / Sm-D2 / snRNP core protein D2
Mass: 12876.066 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein Sm D2 Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) References: UniProt: Q06217
Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ...Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ethane with a Vitrobot Mark III (FEI) operated at 4 degrees Celsius and 100% humidity.
-
Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Average exposure time: 16 sec. / Electron dose: 56 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5115
EM imaging optics
Energyfilter name: GIF Quantum
Image scans
Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 20 / Used frames/image: 1-20
-
Processing
EM software
ID
Name
Version
Category
Details
1
RELION
2
particleselection
2
DigitalMicrograph
imageacquisition
4
CTFFIND
4.1
CTFcorrection
CTFdetermination
5
RELION
2
CTFcorrection
CTFcorrection
8
Coot
0.8.3
modelfitting
9
UCSF Chimera
1.10.2
modelfitting
10
Situs
2.8
modelfitting
12
PHENIX
1.10.1-2155-000
modelrefinement
13
RELION
2
initialEulerassignment
14
RELION
2
finalEulerassignment
15
RELION
2
classification
16
RELION
2
3Dreconstruction
Image processing
Details: Movies were binned once and aligned using MOTIONCORR.
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 496581
Symmetry
Point symmetry: C1 (asymmetric)
3D reconstruction
Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9559 Details: A temperature factor of -170 A2 was applied (sharpened B2 map). Symmetry type: POINT
Atomic model building
B value: 170 / Space: REAL
+
About Yorodumi
-
News
-
Aug 12, 2020. New: Covid-19 info
New: Covid-19 info
New page: Covid-19 featured information page in EM Navigator
Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data
In the structure databanks used in Yorodumi, some data are registered as the other names, "COVID-19 virus" and "2019-nCoV". Here are the details of the virus and the list of structure data.
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)
EMDB accession codes are about to change! (news from PDBe EMDB page)
The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
The EM Navigator/Yorodumi systems omit the EMD- prefix.
Related info.:Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator
wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.
Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi