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- PDB-8vb7: Kinetic intermediate states of HIV-1 RT DNA synthesis captured by... -

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Basic information

Entry
Database: PDB / ID: 8vb7
TitleKinetic intermediate states of HIV-1 RT DNA synthesis captured by cryo-EM
Components
  • DNA (38-MER)
  • HIV-1 reverse transcriptase P51 subunit
  • HIV-1 reverse transcriptase/ribonuclease H P66 subunit
KeywordsTRANSCRIPTION / TRANSFERASE/DNA / Reverse Transcription / Time-resolved / HIV-1 / Cryo-EM / TRANSFERASE-DNA complex
Function / homology
Function and homology information


HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / symbiont-mediated activation of host apoptosis / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase ...HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / symbiont-mediated activation of host apoptosis / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase / viral penetration into host nucleus / symbiont-mediated suppression of host gene expression / RNA stem-loop binding / RNA-directed DNA polymerase activity / 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 / symbiont entry into host cell / viral translational frameshifting / lipid binding / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / 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. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / 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. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / DNA / DNA (> 10) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsVergara, S. / Zhou, X. / Santiago, U. / Conway, J.F. / Sluis-Cremer, N. / Calero, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI175067 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082251 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription.
Authors: Sandra Vergara / Xiaohong Zhou / Ulises Santiago / Mounia Alaoui-El-Azher / James F Conway / Nicolas Sluis-Cremer / Guillermo Calero /
Abstract: Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight ...Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg (site B) inducing formation of a negatively charged pocket where a second floating Mg can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg and the primer 3'-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.
History
DepositionDec 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HIV-1 reverse transcriptase/ribonuclease H P66 subunit
B: HIV-1 reverse transcriptase P51 subunit
F: DNA (38-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,3045
Polymers127,7893
Non-polymers5152
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein HIV-1 reverse transcriptase/ribonuclease H P66 subunit


Mass: 64104.457 Da / Num. of mol.: 1 / Fragment: UNP residues 600-1154 / Mutation: C280S D498N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: gag-pol / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03366
#2: Protein HIV-1 reverse transcriptase P51 subunit


Mass: 51944.691 Da / Num. of mol.: 1 / Fragment: UNP residues 600-1027
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: gag-pol / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03366
#3: DNA chain DNA (38-MER)


Mass: 11739.513 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: DNA a-tamer / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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

ComponentName: Reverse transcriptase/ribonuclease H in complex with DNA aptamer
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.4
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K

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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: 96000 X / Nominal defocus max: 2250 nm / Nominal defocus min: 750 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
9PHENIX1.20.1_4487:model refinement
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 402495 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE

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