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- PDB-8urj: Cryo-EM structure of the HIV-1 nuclear export complex -

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Basic information

Entry
Database: PDB / ID: 8urj
TitleCryo-EM structure of the HIV-1 nuclear export complex
Components
  • (Rev HIV-1) x 2
  • Exportin-1
  • GTP-binding nuclear protein Ran
  • HIV-1 RRE
KeywordsRNA BINDING PROTEIN/RNA / HIV Karyopherin Rev Crm1 / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


HuR (ELAVL1) binds and stabilizes mRNA / annulate lamellae / regulation of proteasomal ubiquitin-dependent protein catabolic process / pre-miRNA export from nucleus / RNA nuclear export complex / snRNA import into nucleus / regulation of centrosome duplication / nuclear export signal receptor activity / Regulation of cholesterol biosynthesis by SREBP (SREBF) / regulation of protein export from nucleus ...HuR (ELAVL1) binds and stabilizes mRNA / annulate lamellae / regulation of proteasomal ubiquitin-dependent protein catabolic process / pre-miRNA export from nucleus / RNA nuclear export complex / snRNA import into nucleus / regulation of centrosome duplication / nuclear export signal receptor activity / Regulation of cholesterol biosynthesis by SREBP (SREBF) / regulation of protein export from nucleus / protein localization to nucleolus / Rev-mediated nuclear export of HIV RNA / Nuclear import of Rev protein / NEP/NS2 Interacts with the Cellular Export Machinery / GTP metabolic process / tRNA processing in the nucleus / Postmitotic nuclear pore complex (NPC) reformation / nucleocytoplasmic transport / MicroRNA (miRNA) biogenesis / DNA metabolic process / Maturation of hRSV A proteins / mitotic sister chromatid segregation / ribosomal large subunit export from nucleus / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / protein localization to nucleus / viral process / Transcriptional and post-translational regulation of MITF-M expression and activity / mRNA export from nucleus / ribosomal subunit export from nucleus / nuclear pore / Cajal body / Cyclin A/B1/B2 associated events during G2/M transition / ribosomal small subunit export from nucleus / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / NPAS4 regulates expression of target genes / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / centriole / Resolution of Sister Chromatid Cohesion / protein export from nucleus / mitotic spindle organization / Downregulation of TGF-beta receptor signaling / Deactivation of the beta-catenin transactivating complex / Transcriptional regulation by small RNAs / RHO GTPases Activate Formins / Heme signaling / MAPK6/MAPK4 signaling / recycling endosome / kinetochore / small GTPase binding / positive regulation of protein import into nucleus / protein import into nucleus / Separation of Sister Chromatids / GDP binding / nuclear envelope / melanosome / positive regulation of protein binding / mitotic cell cycle / ribosome biogenesis / G protein activity / midbody / nuclear membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cadherin binding / protein heterodimerization activity / ribonucleoprotein complex / cell division / intracellular membrane-bounded organelle / GTPase activity / chromatin binding / nucleolus / GTP binding / chromatin / magnesium ion binding / protein-containing complex / RNA binding / extracellular exosome / nucleoplasm / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Exportin-1, repeat 3 / Chromosome region maintenance repeat / Exportin-1, repeat 2 / Chromosome region maintenance or exportin repeat / CRM1 / Exportin repeat 2 / CRM1 / Exportin repeat 3 / CRM1 C terminal / Exportin-1, C-terminal / CRM1 C terminal / Exportin-1/5 ...Exportin-1, repeat 3 / Chromosome region maintenance repeat / Exportin-1, repeat 2 / Chromosome region maintenance or exportin repeat / CRM1 / Exportin repeat 2 / CRM1 / Exportin repeat 3 / CRM1 C terminal / Exportin-1, C-terminal / CRM1 C terminal / Exportin-1/5 / Exportin-1/Importin-beta-like / Exportin 1-like protein / small GTPase Ran family profile. / Ran GTPase / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta N-terminal domain profile. / Importin-beta, N-terminal domain / 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
: / RNA / RNA (> 10) / RNA (> 100) / Exportin-1 / GTP-binding nuclear protein Ran
Similarity search - Component
Biological speciesHomo sapiens (human)
Human immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsSmith, A.M. / Cheng, Y. / Frankel, A.D.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI170792 United States
National Institutes of Health/Office of the DirectorOD020054 United States
National Institutes of Health/Office of the DirectorOD021741 United States
National Institutes of Health/Office of the DirectorOD026881 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: To Be Published
Title: Structure of the HIV-1 RNA Nuclear Export Complex Reveals Crm1 Versatility
Authors: Smith, A.M. / Cheng, Y. / Frankel, A.D. / Li, Y. / Velarde, A.
History
DepositionOct 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 30, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Exportin-1
B: Rev HIV-1
C: Exportin-1
D: GTP-binding nuclear protein Ran
E: Rev HIV-1
H: GTP-binding nuclear protein Ran
G: HIV-1 RRE


Theoretical massNumber of molelcules
Total (without water)422,3077
Polymers422,3077
Non-polymers00
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 Exportin-1 / Chromosome Maintenance 1


Mass: 122267.984 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: XPO1 / Production host: Homo sapiens (human) / Strain (production host): HEK293-F / References: UniProt: O14980
#2: Protein Rev HIV-1


Mass: 10687.159 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Native NES was exchanged with NS2Val NES Native NES: LPPLERLTL Ns2Val NES: TVDEMTKKFGTLTI
Source: (gene. exp.) Human immunodeficiency virus 1 / Strain: HXB3 / Gene: Rev / Production host: Escherichia coli BL21 (bacteria)
#3: Protein GTP-binding nuclear protein Ran / Androgen receptor-associated protein 24 / GTPase Ran / Ras-like protein TC4 / Ras-related nuclear protein


Mass: 20722.076 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Q69L 1-180 / Source: (gene. exp.) Homo sapiens (human) / Gene: RAN, ARA24, OK/SW-cl.81 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P62826, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#4: Protein Rev HIV-1


Mass: 10644.108 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Native NES was exchanged with NS2Val NES Native NES: LPPLERLTL Ns2Val NES: TVDEMTKKFGTLTI
Source: (gene. exp.) Human immunodeficiency virus 1 / Strain: HXB3 / Production host: Escherichia coli BL21 (bacteria)
#5: RNA chain HIV-1 RRE


Mass: 114996.102 Da / Num. of mol.: 1 / Mutation: G262A G269A / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1 / References: GenBank: 328658
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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1HIV-1 Nuclear Export complexCOMPLEX#1-#40MULTIPLE SOURCES
2Crm1COMPLEX#11RECOMBINANTCrm1 forms a dimer
3Ran Q69L 1-180COMPLEX#31RECOMBINANTEach Crm1 subunit binds a Ran molecule
4HIV-1 Rev/RRE RNPCOMPLEX#2, #41RECOMBINANTRRE was generated from T7 polymerase on a linearized plasmid
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.445 MDaNO
220.123 MDaNO
330.02075 MDaNO
440.158 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
52Homo sapiens (human)9606
63Homo sapiens (human)9606
74Human immunodeficiency virus 111676
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
32Homo sapiens (human)9606
43Escherichia coli BL21 (bacteria)511693BL21
54Escherichia coli BL21 (bacteria)511693BL21
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
250 mMPotassium ChlorideKCl1
32 mMbeta-mercaptoethanolHOCH2CH2SH1
42 % (v/v)glycerolC3H8O31
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298.15 K
Details: Washed grid 2x outside of vitrobot; with the second drop of buffer I loaded the tweezers; moved the grid into the chamber and then I blotted.

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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 magnification: 105000 X / Calibrated magnification: 59880 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real imagesDetails
1166GATAN K3 BIOQUANTUM (6k x 4k)624447graphene oxide grids treated with single stranded DNA oligos; tilt angles 0 and 25 degrees
2168GATAN K3 BIOQUANTUM (6k x 4k)322762Graphene oxide grids treated with polyamine; tilt angles 0, 15 and 30 degrees
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategoryDetails
1RELIONparticle selectionpolyamine
2cryoSPARCparticle selectionssDNA
5cryoSPARCCTF correction
6RELIONCTF correction
9UCSF ChimeraXmodel fitting
11RELIONinitial Euler assignment
12cryoSPARCinitial Euler assignment
13RELIONfinal Euler assignment
15RELION3D reconstruction
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11176625
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 260885 / Symmetry type: POINT
Atomic model buildingB value: 325 / Protocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDDetailsInitial refinement model-ID
15DIS

5dis

A5DISACrm11
25DIS

5dis

B5DISBRan Q69L 1-1801
34PMI

4pmi

A4PMIARRE stem IIB mimic2
44PMI

4pmi

B4PMIBRev2
54PMI

4pmi

C4PMICRev2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322505
ELECTRON MICROSCOPYf_angle_d0.75830649
ELECTRON MICROSCOPYf_dihedral_angle_d8.3563254
ELECTRON MICROSCOPYf_chiral_restr0.0533506
ELECTRON MICROSCOPYf_plane_restr0.0063764

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