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- PDB-9d45: Cryo-EM structure of yeast Exportin Msn5 bound to cargo Pho4 and ... -

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Basic information

Entry
Database: PDB / ID: 9d45
TitleCryo-EM structure of yeast Exportin Msn5 bound to cargo Pho4 and RanGTP
Components
  • GTP-binding nuclear protein GSP1/CNR1
  • Phosphate system positive regulatory protein PHO4
  • Protein MSN5
KeywordsTRANSPORT PROTEIN / Karyopherin / Exportin / Nuclear Export
Function / homology
Function and homology information


positive regulation of phosphate metabolic process / regulation of cell cycle phase transition / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of nucleocytoplasmic transport / Postmitotic nuclear pore complex (NPC) reformation / tRNA re-export from nucleus / RNA nuclear export complex / nuclear export signal receptor activity / cellular response to phosphate starvation / RNA export from nucleus ...positive regulation of phosphate metabolic process / regulation of cell cycle phase transition / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of nucleocytoplasmic transport / Postmitotic nuclear pore complex (NPC) reformation / tRNA re-export from nucleus / RNA nuclear export complex / nuclear export signal receptor activity / cellular response to phosphate starvation / 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 / sequence-specific DNA binding / protein dimerization activity / chromatin remodeling / DNA-binding transcription factor activity / GTPase activity / GTP binding / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / 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 / Helix-loop-helix DNA-binding domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. ...Exportin-5, C-terminal domain / Exportin-5 family / Exportin-1/5 / small GTPase Ran family profile. / Ran GTPase / Helix-loop-helix DNA-binding domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / helix loop helix domain / Myc-type, basic helix-loop-helix (bHLH) domain / Myc-type, basic helix-loop-helix (bHLH) domain profile. / Helix-loop-helix DNA-binding domain superfamily / 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 / Phosphate system positive regulatory protein PHO4 / 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
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
Cancer Prevention and Research Institute of Texas (CPRIT)RP220582 United States
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
DepositionAug 12, 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
C: Phosphate system positive regulatory protein PHO4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)187,3965
Polymers186,8483
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: Protein Phosphate system positive regulatory protein PHO4


Mass: 22497.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: In vitro phosphorylated
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PHO4, YFR034C / Production host: Escherichia coli (E. coli) / References: UniProt: P07270
#4: 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
#5: 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 modificationY

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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-#3 / Source: RECOMBINANT
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: FEI TITAN 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
IDNameVersionCategory
1cryoSPARC3particle selection
4cryoSPARC3CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARC3initial Euler assignment
10cryoSPARC3final Euler assignment
12cryoSPARC33D reconstruction
19PHENIX1.20.1_4487:model refinement
20Coot0.9model refinement
21ISOLDEmodel refinement
22UCSF ChimeraXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 102030 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDDetailsSource nameTypeAccession codeChain-IDInitial refinement model-ID
1Msn5 model generated based on PDB entry 5YU7SwissModelin silico model
23M1I

3m1i

AGsp1 from PDB entry 3M1I.PDBexperimental model3M1IA2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211375
ELECTRON MICROSCOPYf_angle_d0.53115432
ELECTRON MICROSCOPYf_dihedral_angle_d7.6241486
ELECTRON MICROSCOPYf_chiral_restr0.0381742
ELECTRON MICROSCOPYf_plane_restr0.0041934

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