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- PDB-6wb0: +3 extended HIV-1 reverse transcriptase initiation complex core (... -

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Basic information

Entry
Database: PDB / ID: 6wb0
Title+3 extended HIV-1 reverse transcriptase initiation complex core (pre-translocation state)
Components
  • HIV-1 viral RNA genome fragment
  • Reverse transcriptase/ribonuclease H
  • reverse transcriptase p51 subunit
  • tRNA lysine 3
KeywordsVIRAL PROTEIN/RNA / reverse transcriptase / RNA / protein-RNA complex / tRNA / polymerase / VIRAL PROTEIN / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


integrase activity / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Minus-strand DNA synthesis / Plus-strand DNA synthesis / Uncoating of the HIV Virion / 2-LTR circle formation / Vpr-mediated nuclear import of PICs / Early Phase of HIV Life Cycle / Integration of provirus ...integrase activity / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Minus-strand DNA synthesis / Plus-strand DNA synthesis / Uncoating of the HIV Virion / 2-LTR circle formation / Vpr-mediated nuclear import of PICs / Early Phase of HIV Life Cycle / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / Binding and entry of HIV virion / viral life cycle / HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / Assembly Of The HIV Virion / Budding and maturation of HIV virion / host multivesicular body / DNA integration / protein processing / 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 / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / peptidase activity / 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-mediated suppression of host gene expression / viral translational frameshifting / lipid binding / symbiont entry into host cell / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / zinc ion binding / identical protein 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 / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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
: / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / RNA / RNA (> 10) / RNA (> 100) / Gag-Pol polyprotein / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsLarsen, K.P. / Jackson, L.N. / Kappel, K. / Zhang, J. / Puglisi, E.V.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082545 United States
CitationJournal: J Mol Biol / Year: 2020
Title: Distinct Conformational States Underlie Pausing during Initiation of HIV-1 Reverse Transcription.
Authors: Kevin P Larsen / Junhong Choi / Lynnette N Jackson / Kalli Kappel / Jingji Zhang / Betty Ha / Dong-Hua Chen / Elisabetta Viani Puglisi /
Abstract: A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have ...A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have identified specific sites along the viral RNA genomic template in which reverse transcriptase (RT) stalls. These stalling points, which occur after the addition of three and five template dNTPs, may serve as checkpoints to regulate the precise timing of HIV-1 reverse transcription following viral entry. Structural studies of reverse transcriptase initiation complexes (RTICs) have revealed unique conformations that may explain the slow rate of incorporation; however, questions remain about the temporal evolution of the complex and features that contribute to strong pausing during initiation. Here we present cryo-electron microscopy and single-molecule characterization of an RTIC after three rounds of dNTP incorporation (+3), the first major pausing point during reverse transcription initiation. Cryo-electron microscopy structures of a +3 extended RTIC reveal conformational heterogeneity within the RTIC core. Three distinct conformations were identified, two of which adopt unique, likely off-pathway, intermediates in the canonical polymerization cycle. Single-molecule Förster resonance energy transfer experiments confirm that the +3 RTIC is more structurally dynamic than earlier-stage RTICs. These alternative conformations were selectively disrupted through structure-guided point mutations to shift single-molecule Förster resonance energy transfer populations back toward the on-pathway conformation. Our results support the hypothesis that conformational heterogeneity within the HIV-1 RTIC during pausing serves as an additional means of regulating HIV-1 replication.
History
DepositionMar 26, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Nov 6, 2024Group: Data collection / Structure summary / Category: em_admin / pdbx_entry_details / Item: _em_admin.last_update
Revision 1.4Nov 13, 2024Group: Data collection / Source and taxonomy / Category: em_admin / em_entity_assembly_naturalsource
Item: _em_admin.last_update / _em_entity_assembly_naturalsource.id

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Structure visualization

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Assembly

Deposited unit
A: Reverse transcriptase/ribonuclease H
B: reverse transcriptase p51 subunit
C: HIV-1 viral RNA genome fragment
D: tRNA lysine 3


Theoretical massNumber of molelcules
Total (without water)175,1214
Polymers175,1214
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, gel filtration, native gel electrophoresis, homology
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Reverse transcriptase/ribonuclease H


Mass: 65558.320 Da / Num. of mol.: 1 / Mutation: Q258C, E478Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Production host: Escherichia coli (E. coli)
References: UniProt: P03366, UniProt: P04585*PLUS, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H
#2: Protein reverse transcriptase p51 subunit


Mass: 51585.293 Da / Num. of mol.: 1 / Mutation: C879S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Production host: Escherichia coli (E. coli) / References: UniProt: P03366, UniProt: P04585*PLUS
#3: RNA chain HIV-1 viral RNA genome fragment


Mass: 32623.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1 / References: GenBank: 60651827
#4: DNA/RNA hybrid tRNA lysine 3


Mass: 25354.061 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
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
1+3 extended HIV-1 reverse transcriptase initiation complex core (pre-translocation state)COMPLEXPeripheral dynamic RNA elements belonging to the tRNA lysine 3 primer and the HIV-1 viral RNA genomic template have been masked out during cryo-EM data processing.all0MULTIPLE SOURCES
2HIV-1 reverse transcriptaseCOMPLEXThe C-terminus of p66 contains an unstructured linker and a six-histidine tag that was cleaved prior to full complex formation.#1-#21RECOMBINANT
3HIV-1 RNA genome fragmentCOMPLEXA fragment of the HIV-1 RNA genome that is 101 nucleotides in length.#31MULTIPLE SOURCES
4tRNA lysine 3COMPLEXChemically synthesized and extended tRNA lysine 3. A modified dG containing an N2-cystamine was placed at position 71 and the sequence was extended on the 3' end by a dC, dT, and ddG to bring its total length to 79 nucleotides.#41MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.175 MDaNO
210.117 MDaNO
310.033 MDaNO
410.025 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Human immunodeficiency virus 111676
22Human immunodeficiency virus 111676
33Human immunodeficiency virus 111676
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22Escherichia coli (E. coli)562
Buffer solutionpH: 8
Details: Full complex was prepared 5-8 hours before freezing. Beta-OG was added just prior to freezing.
Buffer component
IDConc.NameFormulaBuffer-ID
175 mMsodium chlorideNaCl1
210 mMtrisC4H11NO31
36 mMmagnesium chlorideMgCl21
40.2 % w/vbeta-octyl glucoside1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 292 K / Details: 3 microliters applied, 2s pre-blot, 2.5s blot.

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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
Image recordingElectron dose: 77.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264696 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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