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- EMDB-25165: Cryo-EM Structure of the PR-RT components of the HIV-1 Pol Polyprotein -

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Basic information

Entry
Database: EMDB / ID: EMD-25165
TitleCryo-EM Structure of the PR-RT components of the HIV-1 Pol Polyprotein
Map dataFull-map generated by Focussed Classification filtered by 2
Sample
  • Complex: PR-RT portion of HIV-1 Pol
    • Protein or peptide: Gag-Pol polyprotein
KeywordsHIV-1 / Reverse transcriptase / Protease / VIRAL PROTEIN / enzyme
Function / homology
Function and homology information


HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus ...HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus / RNA stem-loop binding / RNA-directed DNA polymerase activity / host cell / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / symbiont-mediated suppression of host gene expression / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / lipid binding / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / zinc ion binding / membrane
Similarity search - Function
Reverse transcriptase connection / Reverse transcriptase connection 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 ...Reverse transcriptase connection / Reverse transcriptase connection 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. / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / 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. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 BH10 / Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsLyumkis D / Passos D
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI136680 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI27690 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54AI150472 United States
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-EM structure of the HIV-1 Pol polyprotein provides insights into virion maturation.
Authors: Jerry Joe E K Harrison / Dario Oliveira Passos / Jessica F Bruhn / Joseph D Bauman / Lynda Tuberty / Jeffrey J DeStefano / Francesc Xavier Ruiz / Dmitry Lyumkis / Eddy Arnold /
Abstract: Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields ...Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo-electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.
History
DepositionOct 19, 2021-
Header (metadata) releaseJul 27, 2022-
Map releaseJul 27, 2022-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25165.png

Downloads & links

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Map

FileDownload / File: emd_25165.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull-map generated by Focussed Classification filtered by 2
Voxel sizeX=Y=Z: 1.015 Å
Density
Contour LevelBy AUTHOR: 0.85
Minimum - Maximum-1.4450061 - 7.185451
Average (Standard dev.)0.006522023 (±0.28871262)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 259.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Full-map generated by Focussed Classification

Fileemd_25165_additional_1.map
AnnotationFull-map generated by Focussed Classification
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_25165_half_map_1.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_25165_half_map_2.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PR-RT portion of HIV-1 Pol

EntireName: PR-RT portion of HIV-1 Pol
Components
  • Complex: PR-RT portion of HIV-1 Pol
    • Protein or peptide: Gag-Pol polyprotein

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Supramolecule #1: PR-RT portion of HIV-1 Pol

SupramoleculeName: PR-RT portion of HIV-1 Pol / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: 3D reconstruction of the HIV-1 Pol polyprotein comprising the PR-RT portion
Source (natural)Organism: Human immunodeficiency virus type 1 BH10
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Gag-Pol polyprotein

MacromoleculeName: Gag-Pol polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Strain: isolate BH10
Molecular weightTheoretical: 119.289867 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKALEVLF QGPMGRDNN SPSEAGADRQ GTVSFNFPQI TLWQRPLVTI KIGGQLKEAL LATGADDTVL EEMSLPGRWK PKMIGGIGGF I KVRQYDQI ...String:
MGSSHHHHHH MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKALEVLF QGPMGRDNN SPSEAGADRQ GTVSFNFPQI TLWQRPLVTI KIGGQLKEAL LATGADDTVL EEMSLPGRWK PKMIGGIGGF I KVRQYDQI LIEICGHKAI GTVLVGPTPV NIIGRNLLTQ IGCTLNFPIS PIETVPVKLK PGMDGPKVKQ WPLTEEKIKA LV EICTEME KEGKISKIGP ENPYNTPVFA IKKKDSTKWR KLVDFRELNK RTQDFWEVQL GIPHPAGLKK KKSVTVLDVG DAY FSVPLD EDFRKYTAFT IPSINNETPG IRYQYNVLPQ GWKGSPAIFQ SSMTKILEPF KKQNPDIVIY QYMDDLYVGS DLEI GQHRT KIEELRQHLL RWGLTTPDKK HQKEPPFLWM GYELHPDKWT VQPIVLPEKD SWTVNDIQKL VGKLNWASQI YPGIK VRQL CKLLRGTKAL TEVIPLTEEA ELELAENREI LKEPVHGVYY DPSKDLIAEI QKQGQGQWTY QIYQEPFKNL KTGKYA RMR GAHTNDVKQL TEAVQKITTE SIVIWGKTPK FKLPIQKETW ETWWTEYWQA TWIPEWEFVN TPPLVKLWYQ LEKEPIV GA ETFYVDGAAN RETKLGKAGY VTNKGRQKVV PLTNTTNQKT ELQAIYLALQ DSGLEVNIVT DSQYALGIIQ AQPDKSES E LVNQIIEQLI KKEKVYLAWV PAHKGIGGNE QVDKLVSAGI RKIDDLDGID KAQDEHEKYH SNWRAMASDF NLPPVVAKE IVASCDKCQL KGEAMHGQVD CSPGIWQLDC THLEGKVILV AVHVASGYIE AEVIPAETGQ ETAYFLLKLA GRWPVKTIHT DNGSNFTSA TVKAACWWAG IKQEFGIPYN PQSQGVVESM NKELKKIIGQ VRDQAEHLKT AVQMAVFIHN FKRKGGIGGY S AGERIVDI IATDIQTKEL QKQITKIQNF RVYYRDSRNP LWKGPAKLLW KGEGAVVIQD NSDIKVVPRR KAKIIRDYGK QM AGDDCVA SRQDED

UniProtKB: Gag-Pol polyprotein

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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

Concentration0.3 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Average electron dose: 0.95 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 27555
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0) / Number images used: 27555
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cisTEM (ver. 1.0)

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Atomic model buiding 1

Initial model(PDB ID:

1dlo

,

3bgr

,

2hb4

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coeficient
Output model

PDB-7sjx:
Cryo-EM Structure of the PR-RT components of the HIV-1 Pol Polyprotein

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