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- EMDB-30640: cryo-EM structure of the DEAH-box helicase Prp2 in complex with i... -

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Basic information

Entry
Database: EMDB / ID: EMD-30640
Titlecryo-EM structure of the DEAH-box helicase Prp2 in complex with its coactivator Spp2
Map dataEM map of the DEAH-box helicase Prp2 in complex with its coactivator Spp2
Sample
  • Complex: Complex of ATPase/helicase Prp2 and its coactivator Spp2
    • Protein or peptide: PRP2 isoform 1
    • Protein or peptide: Pre-mRNA-splicing factor SPP2
Keywordsspliceosome / RNA splicing / Bact complex / DEAH-box ATPase/helicase / Prp2 / Spp2 / SPLICING
Function / homology
Function and homology information


snoRNA splicing / generation of catalytic spliceosome for first transesterification step / ATP-dependent activity, acting on RNA / U2-type catalytic step 1 spliceosome / ATPase activator activity / helicase activity / spliceosomal complex / mRNA splicing, via spliceosome / nucleic acid binding / RNA helicase activity ...snoRNA splicing / generation of catalytic spliceosome for first transesterification step / ATP-dependent activity, acting on RNA / U2-type catalytic step 1 spliceosome / ATPase activator activity / helicase activity / spliceosomal complex / mRNA splicing, via spliceosome / nucleic acid binding / RNA helicase activity / RNA helicase / ATP hydrolysis activity / RNA binding / ATP binding / nucleus / cytoplasm
Similarity search - Function
Pre-mRNA-splicing factor Spp2-like / Spp2/MOS2, G-patch domain / G-patch domain / G-patch domain / glycine rich nucleic binding domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix ...Pre-mRNA-splicing factor Spp2-like / Spp2/MOS2, G-patch domain / G-patch domain / G-patch domain / glycine rich nucleic binding domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Helicase-associated domain / Helicase associated domain (HA2) Add an annotation / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PRP2 isoform 1 / Pre-mRNA-splicing factor SPP2 / Pre-mRNA-splicing factor ATP-dependent RNA helicase-like protein PRP2 / Pre-mRNA-splicing factor SPP2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsBai R / Wan R
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31930059 China
CitationJournal: Science / Year: 2021
Title: Mechanism of spliceosome remodeling by the ATPase/helicase Prp2 and its coactivator Spp2.
Authors: Rui Bai / Ruixue Wan / Chuangye Yan / Qi Jia / Jianlin Lei / Yigong Shi /
Abstract: Spliceosome remodeling, executed by conserved adenosine triphosphatase (ATPase)/helicases including Prp2, enables precursor messenger RNA (pre-mRNA) splicing. However, the structural basis for the ...Spliceosome remodeling, executed by conserved adenosine triphosphatase (ATPase)/helicases including Prp2, enables precursor messenger RNA (pre-mRNA) splicing. However, the structural basis for the function of the ATPase/helicases remains poorly understood. Here, we report atomic structures of Prp2 in isolation, Prp2 complexed with its coactivator Spp2, and Prp2-loaded activated spliceosome and the results of structure-guided biochemical analysis. Prp2 weakly associates with the spliceosome and cannot function without Spp2, which stably associates with Prp2 and anchors on the spliceosome, thus tethering Prp2 to the activated spliceosome and allowing Prp2 to function. Pre-mRNA is loaded into a featured channel between the N and C halves of Prp2, where Leu from the N half and Arg from the C half prevent backward sliding of pre-mRNA toward its 5'-end. Adenosine 5'-triphosphate binding and hydrolysis trigger interdomain movement in Prp2, which drives unidirectional stepwise translocation of pre-mRNA toward its 3'-end. These conserved mechanisms explain the coupling of spliceosome remodeling to pre-mRNA splicing.
History
DepositionOct 26, 2020-
Header (metadata) releaseJan 6, 2021-
Map releaseJan 6, 2021-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7dcr
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30640.png

Downloads & links

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Map

FileDownload / File: emd_30640.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map of the DEAH-box helicase Prp2 in complex with its coactivator Spp2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 300 pix.
= 198.75 Å		0.66 Å/pix.
x 300 pix.
= 198.75 Å		0.66 Å/pix.
x 300 pix.
= 198.75 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.6625 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.05151096 - 0.090821825
Average (Standard dev.)0.000077451245 (±0.0022361535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 198.75 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.66250.66250.6625
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z198.750198.750198.750
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0520.0910.000

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Supplemental data

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Sample components

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Entire : Complex of ATPase/helicase Prp2 and its coactivator Spp2

EntireName: Complex of ATPase/helicase Prp2 and its coactivator Spp2
Components
  • Complex: Complex of ATPase/helicase Prp2 and its coactivator Spp2
    • Protein or peptide: PRP2 isoform 1
    • Protein or peptide: Pre-mRNA-splicing factor SPP2

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Supramolecule #1: Complex of ATPase/helicase Prp2 and its coactivator Spp2

SupramoleculeName: Complex of ATPase/helicase Prp2 and its coactivator Spp2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 119 kDa/nm

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Macromolecule #1: PRP2 isoform 1

MacromoleculeName: PRP2 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 99.947492 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSSITSETGK RRVKRTYEVT RQNDNAVRIE PSSLGEEEDK EAKDKNSALQ LKRSRYDPNK VFSNTNQGPE KNNLKGEQLG SQKKSSKYD EKITSNNELT TKKGLLGDSE NETKYASSNS KFNVEVTHKI KNAKEIDKIN RQRMWEEQQL RNAMAGQSDH P DDITLEGS ...String:
MSSITSETGK RRVKRTYEVT RQNDNAVRIE PSSLGEEEDK EAKDKNSALQ LKRSRYDPNK VFSNTNQGPE KNNLKGEQLG SQKKSSKYD EKITSNNELT TKKGLLGDSE NETKYASSNS KFNVEVTHKI KNAKEIDKIN RQRMWEEQQL RNAMAGQSDH P DDITLEGS DKYDYVFDTD AMIDYTNEED DLLPEEKLQY EARLAQALET EEKRILTIQE ARKLLPVHQY KDELLQEIKK NQ VLIIMGE TGSGKTTQLP QYLVEDGFTD QGKLQIAITQ PRRVAATSVA ARVADEMNVV LGKEVGYQIR FEDKTTPNKT VLK YMTDGM LLREFLTDSK LSKYSCIMID EAHERTLATD ILIGLLKDIL PQRPTLKLLI SSATMNAKKF SEFFDNCPIF NVPG RRYPV DIHYTLQPEA NYIHAAITTI FQIHTTQSLP GDILVFLTGQ EEIERTKTKL EEIMSKLGSR TKQMIITPIY ANLPQ EQQL KIFQPTPENC RKVVLATNIA ETSLTIDGIR YVIDPGFVKE NSYVPSTGMT QLLTVPCSRA SVDQRAGRAG RVGPGK CFR IFTKWSYLHE LELMPKPEIT RTNLSNTVLL LLSLGVTDLI KFPLMDKPSI PTLRKSLENL YILGALNSKG TITRLGK MM CEFPCEPEFA KVLYTAATHE QCQGVLEECL TIVSMLHETP SLFIGQKRDA AASVLSEVES DHILYLEIFN QWRNSKFS R SWCQDHKIQF KTMLRVRNIR NQLFRCSEKV GLVEKNDQAR MKIGNIAGYI NARITRCFIS GFPMNIVQLG PTGYQTMGR SSGGLNVSVH PTSILFVNHK EKAQRPSKYV LYQQLMLTSK EFIRDCLVIP KEEWLIDMVP QIFKDLIDDK TNRGRR

UniProtKB: PRP2 isoform 1

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Macromolecule #2: Pre-mRNA-splicing factor SPP2

MacromoleculeName: Pre-mRNA-splicing factor SPP2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 20.685377 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSKFSLKLGS KTLKKNISKK TKKKNSLQKA NLFDWDDAET ASLSHKPQSK IKIQSIDKFD LDEESSSKKK LVIKLSENAD TKKNDAPLV EYVTEKEYNE VPVEEFGDAL LRGMGWESDS EQDSKGDKTQ SRNKDVSNVS QIHPDGLGIG AKLNKAINVE E ASFMPVVK IDKITGTKVD DDKKNKS

UniProtKB: Pre-mRNA-splicing factor SPP2

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 130302
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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