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- EMDB-48430: SPA of purified HIV-1 CA protein in vitro assembled with IP6 (mat... -

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Basic information

Entry
Database: EMDB / ID: EMD-48430
TitleSPA of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly.
Map dataRefine 3D map of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly. Determined via SPA.
Sample
  • Virus: HIV-1 (virus)
    • Protein or peptide: HIV-1 CA
  • Ligand: Lenacapavir
  • Ligand: INOSITOL HEXAKISPHOSPHATE
KeywordsHIV / Mature / VLP / Lenacapavir / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


viral budding via host ESCRT complex / ISG15 antiviral mechanism / host multivesicular body / viral nucleocapsid / viral translational frameshifting / host cell plasma membrane / host cell nucleus / virion membrane / structural molecule activity / RNA binding / zinc ion binding
Similarity search - Function
Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal ...Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal / 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.
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 BH10 / HIV-1 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRicana CL / Dick RA
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI147890 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54AI170855 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
National Science Foundation (NSF, United States)DMR-1719875 United States
CitationJournal: bioRxiv / Year: 2026
Title: Lenacapavir prevents production of infectious HIV-1 by abrogating immature virus assembly.
Authors: Clifton L Ricaña / Savannah G Brancato / Carolyn M Highland / Fatemeh Ekbataniamiri / Krisztina Ambrus / Juan S Rey / Mia Faerch / Bruce E Torbett / Juan R Perilla / Robert A Dick /
Abstract: The HIV-1 capsid effector Lenacapavir (LEN) acts by disrupting the early (reverse transcription and nuclear entry) and late (assembly and maturation) stages of the viral lifecycle. The early stage ...The HIV-1 capsid effector Lenacapavir (LEN) acts by disrupting the early (reverse transcription and nuclear entry) and late (assembly and maturation) stages of the viral lifecycle. The early stage requires an intact Capsid consisting of a lattice of capsid protein (CA) hexamers and pentamers. Phenylalanine-Glycine (FG) containing nuclear pore proteins interact with Capsid hexamers at their FG pockets, facilitating nuclear entry. Disruption of Capsid lattice stability, or competitive binding to the FG pocket, by LEN blocks infection. Here, we provide insight into the effects of LEN on the late stage. Using a combination of cryo-EM structure determination, assembly, and viral assays, we determined that treatment of producer cells with LEN abrogates the production of infectious virus via multiple mechanisms. Previous studies have shown that HIV-1 produced from LEN treated cells have improperly formed Capsids. However, how LEN interacts with the CA domain of the Gag polyprotein during assembly, prior to maturation, is unclear. Using a viral protease (PR) defective HIV-1 clone, which traps the Gag lattice in an immature state, we found that LEN dramatically remodulates the CA domain. The resulting CA layer was mature-like despite the absence of PR cleavage. We dubbed this unnatural state as "premature" lattice. Proper immature and mature lattice formation requires inositol hexakisphosphate (IP6), but our cryo-EM work revealed a lack of IP6 in the premature lattice. These findings provide insight into the mechanisms by which LEN prevents infectious HIV-1 production.
History
DepositionDec 22, 2024-
Header (metadata) releaseJan 14, 2026-
Map releaseJan 14, 2026-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48430.png

Downloads & links

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Map

FileDownload / File: emd_48430.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefine 3D map of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly. Determined via SPA.
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.0018
Minimum - Maximum-0.003998664 - 0.008762086
Average (Standard dev.)0.000007525749 (±0.00041589414)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 295.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48430_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocess map of purified HIV-1 CA protein in...

Fileemd_48430_additional_1.map
AnnotationPostprocess map of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly. Determined via SPA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refine 3D half2 map of purified HIV-1 CA...

Fileemd_48430_half_map_1.map
AnnotationRefine 3D half2 map of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly. Determined via SPA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refine 3D half1 map of purified HIV-1 CA...

Fileemd_48430_half_map_2.map
AnnotationRefine 3D half1 map of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly. Determined via SPA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HIV-1

EntireName: HIV-1 (virus)
Components
  • Virus: HIV-1 (virus)
    • Protein or peptide: HIV-1 CA
  • Ligand: Lenacapavir
  • Ligand: INOSITOL HEXAKISPHOSPHATE

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Supramolecule #1: HIV-1

SupramoleculeName: HIV-1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly.
NCBI-ID: 11698 / Sci species name: HIV-1 / Sci species strain: BH10 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Virus shellShell ID: 1 / Name: Capsid / Diameter: 1000.0 Å

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Macromolecule #1: HIV-1 CA

MacromoleculeName: HIV-1 CA / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus type 1 BH10 / Strain: BH10
Molecular weightTheoretical: 25.630426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRLHPVHAG PIAPGQMREP RGSDIAGTTS TLQEQIGWMT HNPPIPVGEI YKRWIILGLN KIVRMYSPTS ILDIRQGPKE P FRDYVDRF ...String:
PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRLHPVHAG PIAPGQMREP RGSDIAGTTS TLQEQIGWMT HNPPIPVGEI YKRWIILGLN KIVRMYSPTS ILDIRQGPKE P FRDYVDRF YKTLRAEQAS QEVKNWMTET LLVQNANPDC KTILKALGPG ATLEEMMTAC QGVGGPGHKA RVL

UniProtKB: Gag polyprotein

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Macromolecule #2: Lenacapavir

MacromoleculeName: Lenacapavir / type: ligand / ID: 2 / Number of copies: 3 / Formula: QNG
Molecular weightTheoretical: 968.282 Da

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Macromolecule #3: INOSITOL HEXAKISPHOSPHATE

MacromoleculeName: INOSITOL HEXAKISPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: IHP
Molecular weightTheoretical: 660.035 Da
Chemical component information

ChemComp-IHP:
INOSITOL HEXAKISPHOSPHATE

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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

Concentration13 mg/mL
BufferpH: 6.2
Component:
ConcentrationFormulaName
25.0 mMC6H13NO4SMES
2.0 mMC9H15O6PTCEP
500.0 uMC39H32ClF10N7O5S2Lenacapavir

Details: 25 mM MES, 2mM TCEP, 500 uM LEN
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.027 kPa
Details: Vacuum held for 30 sec before 20 mA were applied for 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 279 K / Instrument: LEICA EM GP
DetailsPurified HIV-1 CASPNC protein in vitro assembled with IP6 (immature morphology). 500 uM LEN was added post assembly.

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 93.15 K / Max: 103.15 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 7510 / Average exposure time: 1.731 sec. / Average electron dose: 50.0 e/Å2
Details: Images were collected in movie-mode with 50 frames per image at a total dose of 50 e-/A2.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 25118581
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-initio
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Number images used: 594567
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
Details: Iterative Bayesian sampling approach via RELION 5.0
Final 3D classificationSoftware - Name: RELION (ver. 5.0)

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Atomic model buiding 1

Initial modelPDB ID:

8g6m


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsMolecular dynamics flexible fitting was done using NAMD targeting cross-correlation coefficients.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-9mnm:
SPA of purified HIV-1 CA protein in vitro assembled with IP6 (mature morphology). 500 uM LEN was added post assembly.

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