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- EMDB-52222: Mature HIV-1 matrix from MA-NC cleavage mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-52222
TitleMature HIV-1 matrix from MA-NC cleavage mutant
Map dataB-factor sharpened map
SampleHIV-1 vector pNL4-3 != Human immunodeficiency virus 1

HIV-1 vector pNL4-3

  • Virus: Human immunodeficiency virus 1
    • Complex: HIV-1 mature matrix
      • Protein or peptide: Gag polyprotein
      • Protein or peptide: Gag polyprotein
KeywordsHIV-1 / HIV / matrix / mature / spacer peptide 2 / SP2 / VIRAL PROTEIN
Function / homology
Function and homology information


viral budding via host ESCRT complex / host multivesicular body / ISG15 antiviral mechanism / viral nucleocapsid / viral translational frameshifting / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding ...viral budding via host ESCRT complex / host multivesicular body / ISG15 antiviral mechanism / viral nucleocapsid / viral translational frameshifting / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / zinc ion binding / membrane
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 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsStacey JCV / Hrebik D / Briggs JAG
Funding support Germany, United States, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)240245660 - SFB 1129 (project 5 HGK, project 6 BM, project 931 21 JAGB) Germany
German Research Foundation (DFG)DFG KR 906/7-1 (HGK) Germany
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32 AI055403 United States
Max Planck Society Germany
CitationJournal: bioRxiv / Year: 2024
Title: The conserved HIV-1 spacer peptide 2 triggers matrix lattice maturation.
Authors: James C V Stacey / Dominik Hrebík / Elizabeth Nand / Snehith Dyavari Shetty / Kun Qu / Marius Boicu / Maria Anders-Össwein / Robert A Dick / Walther Mothes / Hans-Georg Kräusslich / ...Authors: James C V Stacey / Dominik Hrebík / Elizabeth Nand / Snehith Dyavari Shetty / Kun Qu / Marius Boicu / Maria Anders-Össwein / Robert A Dick / Walther Mothes / Hans-Georg Kräusslich / Barbara Müller / John A G Briggs /
Abstract: HIV-1 particles are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious ...HIV-1 particles are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious virions. In immature virus particles, the Gag membrane binding domain, MA, forms a hexameric protein lattice that undergoes structural transition upon cleavage into a distinct, mature MA lattice. The mechanism of MA lattice maturation is unknown. Here we show that released spacer peptide 2 (SP2), a conserved peptide of unknown function situated ~300 residues downstream of MA, binds MA to induce structural maturation. By high-resolution in-virus structure determination of MA, we show that MA does not bind lipid into a side pocket as previously thought, but instead binds SP2 as an integral part of the protein-protein interfaces that stabilise the mature lattice. Analysis of Gag cleavage site mutants showed that SP2 release is required for MA maturation, and we demonstrate that SP2 is sufficient to induce maturation of purified MA on lipid layers in vitro. SP2-triggered MA maturation correlated with faster fusion of virus with target cells. Our results reveal a new, unexpected interaction between two HIV-1 components, provide a high-resolution structure of mature MA, establish the trigger of MA structural maturation, and assign function to the SP2 peptide.
History
DepositionNov 29, 2024-
Header (metadata) releaseDec 18, 2024-
Map releaseDec 18, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52222.png

Downloads & links

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Map

FileDownload / File: emd_52222.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 512 pix.
= 486.4 Å		0.95 Å/pix.
x 512 pix.
= 486.4 Å		0.95 Å/pix.
x 512 pix.
= 486.4 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.164
Minimum - Maximum-0.9977988 - 1.5658903
Average (Standard dev.)-0.002641702 (±0.024921963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 486.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52222_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unsharpened map

Fileemd_52222_additional_1.map
AnnotationUnsharpened map
Projections & Slices
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Histogram

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Half map: Half map 2

Fileemd_52222_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_52222_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Sample components

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Entire : HIV-1 vector pNL4-3

EntireName: HIV-1 vector pNL4-3 (others)
Components
  • Virus: Human immunodeficiency virus 1
    • Complex: HIV-1 mature matrix
      • Protein or peptide: Gag polyprotein
      • Protein or peptide: Gag polyprotein

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Supramolecule #1: Human immunodeficiency virus 1

SupramoleculeName: Human immunodeficiency virus 1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: HEK293T cells were transfected with pcHIV which was expressed and purified.
NCBI-ID: 11676 / Sci species name: Human immunodeficiency virus 1 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48 KDa
Virus shellShell ID: 1 / Name: Gag / Diameter: 1200.0 Å

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Supramolecule #2: HIV-1 mature matrix

SupramoleculeName: HIV-1 mature matrix / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Details: HIV-1 mature matrix as a part of the MA-NC polypeptide, in a complex with SP2
Source (natural)Organism: Human immunodeficiency virus 1

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Macromolecule #1: Gag polyprotein

MacromoleculeName: Gag polyprotein / type: protein_or_peptide / ID: 1 / Details: Matrix protein / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GARASVLSGG ELDKWEKIRL RPGGKKQYKL KHIVWASREL ERFAVNPGLL ETSEGCRQIL GQLQPSLQTG SEELRSLYNT IAVLYCVHQ RIDVKDTKEA LDKIEEEQNK SKKKAQQAAA DTGNNSQVSQ NY

UniProtKB: Gag polyprotein

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Macromolecule #2: Gag polyprotein

MacromoleculeName: Gag polyprotein / type: protein_or_peptide / ID: 2 / Details: SP2 peptide / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
FLGKIWPSHK GRPGNF

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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: 7.4
Component:
ConcentrationFormulaName
8.1 mmol/LNa2HPO4disodium hydrogen phosphate
1.5 mmol/LKH2PO4potassium dihydrogen phosphate
137.0 mmol/LNaClsodium chloride
2.7 mmol/LKClpotassium chloride

Details: PBS
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: LEICA EM GP
Detailspurified HIV-1 MA-NC particles in PBS

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 88.0 K / Max: 93.0 K
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 15 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 17335 / Average exposure time: 4.0 sec. / Average electron dose: 40.0 e/Å2 / Details: EER mode
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 0.48 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
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: 7238540
Details: A new model was trained in crYOLO using a training dataset annotated in a randomly selected set of 50-100 micrographs. Annotation was performed in the crYOLO boxmanager GUI placing positions ...Details: A new model was trained in crYOLO using a training dataset annotated in a randomly selected set of 50-100 micrographs. Annotation was performed in the crYOLO boxmanager GUI placing positions all over the visible surface of an HIV virus particle. The picks did not distinguish individual proteins or membranes.
Startup modelType of model: EMDB MAP
EMDB ID:

13088

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3) / Number images used: 7745404
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3)
Final 3D classificationNumber classes: 3 / Avg.num./class: 364808 / Software - Name: cryoSPARC (ver. 4.3)
Details: particles were symmetry expanded and moved to the neighbouring symmetry centres after classification
FSC plot (resolution estimation)

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