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- EMDB-60685: Structure of influenza A virus RNA polymerase PB1 and nuclear imp... -

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Basic information

Entry
Database: EMDB / ID: EMD-60685
TitleStructure of influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex
Map data
Sample
  • Complex: influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex.
    • Complex: RanBP5
      • Protein or peptide: Importin-5
    • Complex: RNA polymerase PB1
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
Keywordsvirus RNA polymerase / NUCLEAR PROTEIN
Function / homology
Function and homology information


cRNA Synthesis / Influenza Infection / Fusion of the Influenza Virion to the Host Cell Endosome / Release / Budding / Packaging of Eight RNA Segments / Uncoating of the Influenza Virion / Entry of Influenza Virion into Host Cell via Endocytosis / GTPase inhibitor activity / vRNA Synthesis ...cRNA Synthesis / Influenza Infection / Fusion of the Influenza Virion to the Host Cell Endosome / Release / Budding / Packaging of Eight RNA Segments / Uncoating of the Influenza Virion / Entry of Influenza Virion into Host Cell via Endocytosis / GTPase inhibitor activity / vRNA Synthesis / Viral RNP Complexes in the Host Cell Nucleus / Transport of Ribonucleoproteins into the Host Nucleus / NLS-bearing protein import into nucleus / nuclear localization sequence binding / ribosomal protein import into nucleus / NEP/NS2 Interacts with the Cellular Export Machinery / nuclear import signal receptor activity / Viral Messenger RNA Synthesis / vRNP Assembly / cytoplasmic pattern recognition receptor signaling pathway / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / negative regulation of cell cycle / Viral mRNA Translation / nuclear pore / cellular response to amino acid stimulus / small GTPase binding / positive regulation of protein import into nucleus / protein import into nucleus / nuclear membrane / host cell cytoplasm / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed RNA polymerase activity / nucleotide binding / DNA-templated transcription / nucleolus / host cell nucleus / Golgi apparatus / RNA binding / extracellular region / nucleoplasm / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Importin repeat / Importin repeat 6 / Importin repeat 4 / Importin repeat / Importin repeat / Importin repeat 6 / Importin beta family / TOG domain / TOG / HEAT-like repeat ...Importin repeat / Importin repeat 6 / Importin repeat 4 / Importin repeat / Importin repeat / Importin repeat 6 / Importin beta family / TOG domain / TOG / HEAT-like repeat / HEAT repeat / HEAT repeat / Importin-beta N-terminal domain profile. / Importin-beta, N-terminal domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Importin-5 / RNA-directed RNA polymerase catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human) / Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) / Influenza A virus (strain A/Puerto Rico/8/1934 H1N1)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsUchikubo-Kamo T / Ishimoto N / Park SY
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Biochem Biophys Res Commun / Year: 2024
Title: Structural insights into Influenza A virus RNA polymerase PB1 binding to nuclear import host factor RanBP5.
Authors: Tomomi Uchikubo-Kamo / Naito Ishimoto / Haruka Umezawa / Mikako Hirohama / Maasa Ono / Haruka Kawabata / Kenichi Kamata / Mio Ohki / Hisashi Yoshida / Jae-Hyun Park / Jeremy R H Tame / ...Authors: Tomomi Uchikubo-Kamo / Naito Ishimoto / Haruka Umezawa / Mikako Hirohama / Maasa Ono / Haruka Kawabata / Kenichi Kamata / Mio Ohki / Hisashi Yoshida / Jae-Hyun Park / Jeremy R H Tame / Atsushi Kawaguchi / Sam-Yong Park /
Abstract: The genome of influenza A viruses consists of eight RNA segments that form a heterotrimer, and the viral genome undergoes transcription and replication in the nucleus. Thus, during infection, newly ...The genome of influenza A viruses consists of eight RNA segments that form a heterotrimer, and the viral genome undergoes transcription and replication in the nucleus. Thus, during infection, newly synthesized RNA polymerase subunits must be imported into the nucleus. Although several models have been proposed for this process, the consensus is that the RNA polymerase subunits PB1 and PA form a dimer in the cytoplasm and are transported into the nucleus by Ran binding protein 5 (RanBP5). The PB2 subunit undergoes separate transport to complete the nuclear import. However, the molecular mechanism of nuclear import by host factors and their interactions with proteins are largely unknown. Here we present the structural analysis of the RanBP5 and PB1 NLS domain complex by cryo-EM at 3.2 Å resolution. The pattern shows that the NLS domain of PB1 does not exist in a secondary structure and interacts with RanBP5 in a wrapped state. In addition, biochemical analyses of the mutant have identified critical amino acid sites involved in complex binding. The results suggest a stepwise assembly of influenza virus structural components regulated by nuclear import mechanisms and host factor binding, with important implications for drug discovery research.
History
DepositionJul 2, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60685.png

Downloads & links

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Map

FileDownload / File: emd_60685.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.35700458 - 0.62977946
Average (Standard dev.)0.0002680041 (±0.014965555)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60685_msk_1.map
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Additional map: #1

Fileemd_60685_additional_1.map
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Half map: #2

Fileemd_60685_half_map_1.map
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Half map: #1

Fileemd_60685_half_map_2.map
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Sample components

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Entire : influenza A virus RNA polymerase PB1 and nuclear import host fact...

EntireName: influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex.
Components
  • Complex: influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex.
    • Complex: RanBP5
      • Protein or peptide: Importin-5
    • Complex: RNA polymerase PB1
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit

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Supramolecule #1: influenza A virus RNA polymerase PB1 and nuclear import host fact...

SupramoleculeName: influenza A virus RNA polymerase PB1 and nuclear import host factor RanBP5 complex.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: RanBP5

SupramoleculeName: RanBP5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: RNA polymerase PB1

SupramoleculeName: RNA polymerase PB1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Influenza A virus (strain A/Puerto Rico/8/1934 H1N1)

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Macromolecule #1: Importin-5

MacromoleculeName: Importin-5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 126.154023 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRGSHHHHHH GSENLYFQGG SMAAAAAEQQ QFYLLLGNLL SPDNVVRKQA EETYENIPGQ SKITFLLQAI RNTTAAEEAR QMAAVLLRR LLSSAFDEVY PALPSDVQTA IKSELLMIIQ METQSSMRKK VCDIAAELAR NLIDEDGNNQ WPEGLKFLFD S VSSQNVGL ...String:
MRGSHHHHHH GSENLYFQGG SMAAAAAEQQ QFYLLLGNLL SPDNVVRKQA EETYENIPGQ SKITFLLQAI RNTTAAEEAR QMAAVLLRR LLSSAFDEVY PALPSDVQTA IKSELLMIIQ METQSSMRKK VCDIAAELAR NLIDEDGNNQ WPEGLKFLFD S VSSQNVGL REAALHIFWN FPGIFGNQQQ HYLDVIKRML VQCMQDQEHP SIRTLSARAT AAFILANEHN VALFKHFADL LP GFLQAVN DSCYQNDDSV LKSLVEIADT VPKYLRPHLE ATLQLSLKLC GDTSLNNMQR QLALEVIVTL SETAAAMLRK HTN IVAQTI PQMLAMMVDL EEDEDWANAD ELEDDDFDSN AVAGESALDR MACGLGGKLV LPMIKEHIMQ MLQNPDWKYR HAGL MALSA IGEGCHQQME GILNEIVNFV LLFLQDPHPR VRYAACNAVG QMATDFAPGF QKKFHEKVIA ALLQTMEDQG NQRVQ AHAA AALINFTEDC PKSLLIPYLD NLVKHLHSIM VLKLQELIQK GTKLVLEQVV TSIASVADTA EEKFVPYYDL FMPSLK HIV ENAVQKELRL LRGKTIECIS LIGLAVGKEK FMQDASDVMQ LLLKTQTDFN DMEDDDPQIS YMISAWARMC KILGKEF QQ YLPVVMGPLM KTASIKPEVA LLDTQDMENM SDDDGWEFVN LGDQQSFGIK TAGLEEKSTA CQMLVCYAKE LKEGFVEY T EQVVKLMVPL LKFYFHDGVR VAAAESMPLL LECARVRGPE YLTQMWHFMC DALIKAIGTE PDSDVLSEIM HSFAKCIEV MGDGCLNNEH FEELGGILKA KLEEHFKNQE LRQVKRQDED YDEQVEESLQ DEDDNDVYIL TKVSDILHSI FSSYKEKVLP WFEQLLPLI VNLICPHRPW PDRQWGLCIF DDVIEHCSPA SFKYAEYFLR PMLQYVCDNS PEVRQAAAYG LGVMAQYGGD N YRPFCTEA LPLLVRVIQS ADSKTKENVN ATENCISAVG KIMKFKPDCV NVEEVLPHWL SWLPLHEDKE EAVQTFNYLC DL IESNHPI VLGPNNTNLP KIFSIIAEGE MHEAIKHEDP CAKRLANVVR QVQTSGGLWT ECIAQLSPEQ QAAIQELLNS A

UniProtKB: Importin-5

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Macromolecule #2: RNA-directed RNA polymerase catalytic subunit

MacromoleculeName: RNA-directed RNA polymerase catalytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Influenza A virus (strain A/Puerto Rico/8/1934 H1N1)
Molecular weightTheoretical: 6.51849 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HHHHHHGSEN LYFQGGSTTH FQRKRRVRDN MTKKMITQRT IGKKKQRLNK RSY

UniProtKB: RNA-directed RNA polymerase catalytic subunit

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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: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeTFS KRIOS
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Average electron dose: 47.37 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Average electron dose: 47.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6xu2

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 226937
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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