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- PDB-9cnv: HIV-2 CA hexamer bound with CPSF6 peptide; assembled via liposome... -

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Basic information

Entry
Database: PDB / ID: 9cnv
TitleHIV-2 CA hexamer bound with CPSF6 peptide; assembled via liposome templating
Components
  • Capsid protein p24
  • Isoform 2 of Cleavage and polyadenylation specificity factor subunit 6
KeywordsVIRAL PROTEIN / HIV-2 / Capsid / IP6 / CPSF6
Function / homology
Function and homology information


exon-exon junction complex binding / HIV-2 retropepsin / positive regulation of RNA export from nucleus / mRNA cleavage factor complex / co-transcriptional mRNA 3'-end processing, cleavage and polyadenylation pathway / interchromatin granule / perichromatin fibrils / Processing of Intronless Pre-mRNAs / mRNA cleavage and polyadenylation specificity factor complex / mRNA alternative polyadenylation ...exon-exon junction complex binding / HIV-2 retropepsin / positive regulation of RNA export from nucleus / mRNA cleavage factor complex / co-transcriptional mRNA 3'-end processing, cleavage and polyadenylation pathway / interchromatin granule / perichromatin fibrils / Processing of Intronless Pre-mRNAs / mRNA cleavage and polyadenylation specificity factor complex / mRNA alternative polyadenylation / mRNA 3'-end processing / Signaling by cytosolic FGFR1 fusion mutants / mRNA 3'-end processing / paraspeckles / RNA Polymerase II Transcription Termination / protein heterotetramerization / ribosomal large subunit binding / Processing of Capped Intron-Containing Pre-mRNA / Signaling by FGFR1 in disease / protein tetramerization / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / viral penetration into host nucleus / host multivesicular body / RNA-directed DNA polymerase activity / mRNA processing / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / host cell / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / nuclear speck / ribonucleoprotein complex / symbiont-mediated suppression of host gene expression / viral translational frameshifting / mRNA binding / symbiont entry into host cell / lipid binding / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / RNA binding / zinc ion binding / nucleoplasm / nucleus / membrane / cytoplasm
Similarity search - Function
Cleavage and polyadenylation specificity factor subunit 6 / CPSF6/7 family / gag protein p24 N-terminal domain / RNA recognition motif / RNA recognition motif / Reverse transcriptase connection / Reverse transcriptase connection domain / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / Reverse transcriptase thumb ...Cleavage and polyadenylation specificity factor subunit 6 / CPSF6/7 family / gag protein p24 N-terminal domain / RNA recognition motif / RNA recognition motif / Reverse transcriptase connection / Reverse transcriptase connection domain / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / RNA-binding domain superfamily / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / RNase H type-1 domain profile. / Ribonuclease H domain / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / Gag-Pol polyprotein / Cleavage and polyadenylation specificity factor subunit 6
Similarity search - Component
Biological speciesHuman immunodeficiency virus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsCook, M. / Freniere, C. / Xiong, Y.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32GM008283 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54AI170791 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P50AI150481 United States
CitationJournal: Cell Rep / Year: 2025
Title: Structural insights into HIV-2 CA lattice formation and FG-pocket binding revealed by single-particle cryo-EM.
Authors: Matthew Cook / Christian Freniere / Chunxiang Wu / Faith Lozano / Yong Xiong /
Abstract: One of the striking features of human immunodeficiency virus (HIV) is the capsid, a fullerene cone comprised of pleomorphic capsid protein (CA) that shields the viral genome and recruits cofactors. ...One of the striking features of human immunodeficiency virus (HIV) is the capsid, a fullerene cone comprised of pleomorphic capsid protein (CA) that shields the viral genome and recruits cofactors. Despite significant advances in understanding the mechanisms of HIV-1 CA assembly and host factor interactions, HIV-2 CA assembly remains poorly understood. By templating the assembly of HIV-2 CA on functionalized liposomes, we report high-resolution structures of the HIV-2 CA lattice, including both CA hexamers and pentamers, alone and with peptides of host phenylalanine-glycine (FG)-motif proteins Nup153 and CPSF6. While the overall fold and mode of FG-peptide binding is conserved with HIV-1, this study reveals distinctive features of the HIV-2 CA lattice, including differing structural character at regions of host factor interactions and divergence in the mechanism of formation of CA hexamers and pentamers. This study extends our understanding of HIV capsids and highlights an approach facilitating the study of lentiviral capsid biology.
History
DepositionJul 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 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: Capsid protein p24
B: Isoform 2 of Cleavage and polyadenylation specificity factor subunit 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,6804
Polymers28,3602
Non-polymers1,3202
Water00
1
A: Capsid protein p24
B: Isoform 2 of Cleavage and polyadenylation specificity factor subunit 6
hetero molecules
x 6


  • complete point assembly
  • Evidence: electron microscopy, HIV-2 CA hexamers are apparent from the patterned assembly on CLPs and appear readily from 2D class averages. This characteristic and binding with CPSF6 peptide are ...Evidence: electron microscopy, HIV-2 CA hexamers are apparent from the patterned assembly on CLPs and appear readily from 2D class averages. This characteristic and binding with CPSF6 peptide are consistent with HIV-1 CA behavior.
  • 178 kDa, 12 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)178,08224
Polymers170,16212
Non-polymers7,92012
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryPoint symmetry: (Schoenflies symbol: C1 (asymmetric))

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Components

#1: Protein Capsid protein p24 / CA


Mass: 26809.490 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: HIV-2 capsid protein with C-terminal Gly-Ser-Ser linker to hexahistidine tag following proteolytic processing of the N-terminal Met.
Source: (gene. exp.) Human immunodeficiency virus 2 / Strain: GL-AN / Gene: gag-pol / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P18042
#2: Protein/peptide Isoform 2 of Cleavage and polyadenylation specificity factor subunit 6 / Cleavage and polyadenylation specificity factor 68 kDa subunit / CPSF 68 kDa subunit / Cleavage ...Cleavage and polyadenylation specificity factor 68 kDa subunit / CPSF 68 kDa subunit / Cleavage factor Im complex 68 kDa subunit / CFIm68 / Pre-mRNA cleavage factor Im 68 kDa subunit / Protein HPBRII-4/7


Mass: 1550.796 Da / Num. of mol.: 1 / Mutation: Delta(1-312) and Delta(328-358) / Source method: obtained synthetically / Details: Truncation peptide of CPSF6 (313-327) / Source: (synth.) Homo sapiens (human) / References: UniProt: Q16630
#3: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H18O24P6
Has ligand of interestN
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
IDNameTypeDetailsEntity IDParent-IDSource
1HIV-2 capsid protein assembled into a lattice via liposome templating and then bound with CPSF6 peptide.COMPLEXC-terminally hexahistidine tagged HIV-2 CA associated with a liposome decorated with NiNTA headgroups which results in the assembly of a lattice of CA. CPSF6 peptide was then introduced and allowed to equilibrate binding.#1-#20MULTIPLE SOURCES
2HIV-2 capsid proteinCOMPLEX#11RECOMBINANT
3CPSF6 peptideCOMPLEX#21SYNTHETIC
Molecular weightValue: 0.0269 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Human immunodeficiency virus 211709GL-AN
33Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
Details: The mixed buffer of storage buffer for the protein and lipid components with IP6 supplemented.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2250 mMSodium chlorideNaCl1
30.1 mMTCEP1
44 mMIP61
SpecimenConc.: 10.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was prepared with 400 uM HIV-2 CA-6xHis protein, 5.9 mM lipid mix (described in publication), and 4 mM IP6 as final concentrations following subsequent addition of CPSF6 peptide. ...Details: Sample was prepared with 400 uM HIV-2 CA-6xHis protein, 5.9 mM lipid mix (described in publication), and 4 mM IP6 as final concentrations following subsequent addition of CPSF6 peptide. After assembly, CPSF6 was added to a final concentration of 400 uM. Sample was well-distributed on the grid, mostly monodisperse. Perhaps slightly more particles on carbon versus in the hole.
Specimen supportDetails: 15 mA discharge current. / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: Grids were dual-side blotted with blot force 0 for 5.5 sec before plunge freezing in liquid 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 magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 15 eV

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selection
2EPUimage acquisitionCollected at Brookhaven National Laboratories
4cryoSPARCCTF correction
7Cootmodel fitting
9Cootmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 23033474
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2537344 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: The HIV-2 CA pentamer chain derived from micelle-templated icosahedra described in this publication was used as an initial model for fitting. Flexible fitting was used to move the CTD into ...Details: The HIV-2 CA pentamer chain derived from micelle-templated icosahedra described in this publication was used as an initial model for fitting. Flexible fitting was used to move the CTD into its proper location. Backbone tracing was used to model the CPSF6 peptide.
Atomic model buildingAccession code: 9CLJ / Chain residue range: 1-223
Details: NTDs matched well, but the CTD had to be realigned.
Source name: Other / Type: experimental model

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