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- PDB-9dz6: Cryo-EM structure of yeast Exportin Msn5 bound to RanGTP and Pho4... -

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Basic information

Entry
Database: PDB / ID: 9dz6
TitleCryo-EM structure of yeast Exportin Msn5 bound to RanGTP and Pho4 (not modeled) (State 3-1)
Components
  • GTP-binding nuclear protein GSP1/CNR1
  • Protein MSN5
KeywordsTRANSPORT PROTEIN / Karyopherin / Exportin / Nuclear Export
Function / homology
Function and homology information


regulation of cell cycle phase transition / regulation of nucleocytoplasmic transport / Postmitotic nuclear pore complex (NPC) reformation / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / tRNA re-export from nucleus / RNA nuclear export complex / nuclear export signal receptor activity / RNA export from nucleus / poly(A)+ mRNA export from nucleus / nucleus organization ...regulation of cell cycle phase transition / regulation of nucleocytoplasmic transport / Postmitotic nuclear pore complex (NPC) reformation / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / tRNA re-export from nucleus / RNA nuclear export complex / nuclear export signal receptor activity / RNA export from nucleus / poly(A)+ mRNA export from nucleus / nucleus organization / ribosomal subunit export from nucleus / protein export from nucleus / protein import into nucleus / GTPase activity / GTP binding / RNA binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Exportin-5, C-terminal domain / Exportin-5 family / Exportin-1/5 / small GTPase Ran family profile. / Ran GTPase / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family ...Exportin-5, C-terminal domain / Exportin-5 family / Exportin-1/5 / small GTPase Ran family profile. / Ran GTPase / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / GTP-binding nuclear protein GSP1/CNR1 / Protein MSN5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsFung, H.Y.J. / Chook, Y.M.
Funding support United States, 5items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of Texas (CPRIT)RP220582 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141461 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM069909 United States
Welch FoundationI-1532 United States
Other privateUTSW Gilman Special Opportunities Award
CitationJournal: Nat Commun / Year: 2025
Title: Phosphate-dependent nuclear export via a non-classical NES class recognized by exportin Msn5.
Authors: Ho Yee Joyce Fung / Sanraj R Mittal / Ashley B Niesman / Jenny Jiou / Binita Shakya / Takuya Yoshizawa / Ahmet E Cansizoglu / Michael P Rout / Yuh Min Chook /
Abstract: Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the ...Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Here, we present a high resolution cryogenic-electron microscopy structure showing the phosphorylated 35-residue nuclear export signal of Pho4, which binds the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches. These findings characterize a mechanism of phosphate-specific recognition mediated by a non-classical signal distinct from that for Exportin-1. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4's release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.
History
DepositionOct 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 2, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein MSN5
B: GTP-binding nuclear protein GSP1/CNR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,8984
Polymers164,3512
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein MSN5


Mass: 143067.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MSN5, YDR335W, D9651.5 / Production host: Escherichia coli (E. coli) / References: UniProt: P52918
#2: Protein GTP-binding nuclear protein GSP1/CNR1 / Chromosome stability protein 17 / GTPase Ran homolog / Genetic suppressor of PRP20-1


Mass: 21283.520 Da / Num. of mol.: 1 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: GSP1, CNR1, CST17, YLR293C, L8003.19 / Production host: Escherichia coli (E. coli) / References: UniProt: P32835
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Msn5-Gsp1-pPho4(1-200) / Type: COMPLEX
Details: Msn5 bound to RanGTP (Gsp1) and phosphorylated Pho4 fragment (1-200)
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryFitting-ID
1cryoSPARC3particle selection
3SerialEMimage acquisition
5cryoSPARC3CTF correction
8UCSF ChimeraXmodel fitting1
10PHENIX1.21-5207model refinement1
11Coot0.9model refinement1
12ISOLDEmodel refinement1
13UCSF ChimeraXmodel refinement1
18cryoSPARC3initial Euler assignment
20cryoSPARC3final Euler assignment
23cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112923 / Symmetry type: POINT
Atomic model building
ID
1
2
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeChain-IDDetailsInitial refinement model-IDSource nameType
13M1I

3m1i

A13M1IAGsp1 from PDB entry 3M1I1PDBexperimental model
21Msn5 model generated based on PDB entry 5YU7SwissModelin silico model
32
42
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410315
ELECTRON MICROSCOPYf_angle_d0.44713981
ELECTRON MICROSCOPYf_dihedral_angle_d11.9113838
ELECTRON MICROSCOPYf_chiral_restr0.0361589
ELECTRON MICROSCOPYf_plane_restr0.0031740

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