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- PDB-6b19: Architecture of HIV-1 reverse transcriptase initiation complex core -

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Basic information

Entry
Database: PDB / ID: 6b19
TitleArchitecture of HIV-1 reverse transcriptase initiation complex core
Components
  • RNA genome fragment
  • reverse transcriptase p51 subunit
  • reverse transcriptase p66 subunit
  • tRNA lysine3
KeywordsVIRAL PROTEIN/RNA / Reverse Transcriptase / tRNA / HIV-1 / Reverse Transcription / RNA / Transcription / Complex / RNA-binding protein / backbone model / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA ...HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / viral penetration into host nucleus / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / lipid binding / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / proteolysis / DNA binding / RNA 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. / Ribonuclease H superfamily / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain 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
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLarsen, K.P. / Mathiharan, Y.K. / Chen, D.H. / Puglisi, J.D. / Skiniotis, G. / 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: Nature / Year: 2018
Title: Architecture of an HIV-1 reverse transcriptase initiation complex.
Authors: Kevin P Larsen / Yamuna Kalyani Mathiharan / Kalli Kappel / Aaron T Coey / Dong-Hua Chen / Daniel Barrero / Lauren Madigan / Joseph D Puglisi / Georgios Skiniotis / Elisabetta Viani Puglisi /
Abstract: Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection and a common target of antiretroviral drugs . The reaction is catalysed by viral reverse ...Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection and a common target of antiretroviral drugs . The reaction is catalysed by viral reverse transcriptase (RT) that is packaged in an infectious virion with two copies of viral genomic RNA each bound to host lysine 3 transfer RNA (tRNA), which acts as a primer for initiation of reverse transcription. Upon viral entry into cells, initiation is slow and non-processive compared to elongation. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNA and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.
History
DepositionSep 18, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 25, 2018Provider: repository / Type: Initial release
Revision 1.1May 9, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: reverse transcriptase p66 subunit
B: reverse transcriptase p51 subunit
C: RNA genome fragment
D: tRNA lysine3


Theoretical massNumber of molelcules
Total (without water)174,4884
Polymers174,4884
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology, Fitting of other HIV reverse transcriptase crystal structures validated our identification of the reverse transcriptase subunits, tRNA primer strand, and viral RNA template strand. ...Evidence: homology, Fitting of other HIV reverse transcriptase crystal structures validated our identification of the reverse transcriptase subunits, tRNA primer strand, and viral RNA template strand., cross-linking, tRNA has been crosslinked from position G71 to 258C on the p66 subunit of HIV reverse transcriptase. This helped validate the position of the viral RNA/tRNA duplex in the cleft.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein reverse transcriptase p66 subunit


Mass: 65558.320 Da / Num. of mol.: 1 / Fragment: UNP residues 600-1159 / Mutation: Q260C, C282S, E480Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Production host: Escherichia coli (E. coli) / References: UniProt: P03366, RNA-directed DNA polymerase
#2: Protein reverse transcriptase p51 subunit


Mass: 51585.293 Da / Num. of mol.: 1 / Fragment: UNP residues 600-1039 / Mutation: C282S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Production host: Escherichia coli (E. coli) / References: UniProt: P03366, RNA-directed DNA polymerase
#3: RNA chain 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 lysine3


Mass: 24720.664 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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
1HIV-1 reverse transcription initiation complex coreCOMPLEXPeripheral dynamic RNA elements belonging to tRNA primer and viral RNA template have been masked out for higher resolution structure determination.all0MULTIPLE SOURCES
2HIV-1 reverse transcriptaseReverse transcriptaseCOMPLEXThe COOH-terminus of p66 contains an unstructured linker and a six-histidine tag that was cleaved before full complex formation. A cysteine mutation for crosslinking was introduced into helix H of p66 (Q258C). The protein used in this study also had the C280S mutation, introduced in prior structural work and the E478Q mutation, introduced to eliminate RNase H activity.#1-#21RECOMBINANT
3HIV-1 RNA genome fragmentCOMPLEXHIV-1 RNA genome fragment. 101 bases in length before masking. Contains the primer binding site (PBS), primer activation signal (PAS), A-rich loop, and C-rich region.#31NATURAL
4tRNA lysine3 primerCOMPLEXChemically synthesized and extended tRNA lysine3 primer. A modified nucleotide containing a N2-cystamine was placed at position 71. The tRNA primer has been extended by one ddCTP, bringing its total length in the full complex to 77 nucleotides before masking.#41NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.175 MDaNO
220.117 MDaNO
330.033 MDaNO
440.025 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Human immunodeficiency virus 111676
23Human immunodeficiency virus 111676
34Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: Beta-OG was added just prior to freezing.
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaClSodium chloride1
210 mMTris-HClTrisC4H11NO31
30.25 % w/vbeta-octyl glucoside1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 292 K
Details: Blotted for 3.5 sec before plunging into liquid ethane.

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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 FIELDBright-field microscopy / Nominal magnification: 29000 X / Calibrated magnification: 50000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4209
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameCategory
4CTFFIND4CTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 765688
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128153 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL / Details: Main chain backbone for protein

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