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- PDB-7kjv: Structure of HIV-1 reverse transcriptase initiation complex core -

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Basic information

Entry
Database: PDB / ID: 7kjv
TitleStructure of HIV-1 reverse transcriptase initiation complex core
Components
  • HIV-1 viral RNA fragment
  • Reverse transcriptase/ribonuclease H
  • reverse transcriptase p51 subunit
  • tRNA lysine 3 fragment with GNRA tetraloop
Keywordsviral protein/RNA / reverse transcriptase / RNA / protein-RNA complex / tRNA / polymerase / viral protein-RNA complex / VIRAL PROTEIN
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 ...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-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) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus type 1 group M subtype B
Human immunodeficiency virus 1
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHa, B. / Larsen, K.P. / Zhang, J. / Fu, Z. / Montabana, E. / Jackson, L.N. / Chen, D.H. / Puglisi, E.V.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI150464 United States
CitationJournal: Nat Commun / Year: 2021
Title: High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs.
Authors: Betty Ha / Kevin P Larsen / Jingji Zhang / Ziao Fu / Elizabeth Montabana / Lynnette N Jackson / Dong-Hua Chen / Elisabetta Viani Puglisi /
Abstract: Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNA primer bound to the ...Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNA primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNA initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.
History
DepositionOct 26, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1May 12, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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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 fragment
D: tRNA lysine 3 fragment with GNRA tetraloop
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,6406
Polymers137,3234
Non-polymers3172
Water0
1


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

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Reverse transcriptase/ribonuclease H / Pr160Gag-Pol


Mass: 64705.316 Da / Num. of mol.: 1 / Mutation: Q258C, E478Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Strain: isolate BH10 / Gene: gag-pol / Production host: Escherichia coli (E. coli)
References: UniProt: P03366, 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
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Strain: isolate BH10 / Gene: gag-pol / Production host: Escherichia coli (E. coli) / References: UniProt: P03366

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RNA chain / DNA/RNA hybrid / Non-polymers / Sugars , 4 types, 4 molecules CD

#3: RNA chain HIV-1 viral RNA fragment


Mass: 8437.105 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1
#4: DNA/RNA hybrid tRNA lysine 3 fragment with GNRA tetraloop


Mass: 12595.636 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Sugar ChemComp-BOG / octyl beta-D-glucopyranoside / Beta-Octylglucoside / octyl beta-D-glucoside / octyl D-glucoside / octyl glucoside / Octyl glucoside


Type: D-saccharide / Mass: 292.369 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H28O6 / Comment: detergent*YM
IdentifierTypeProgram
b-octylglucosideIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0

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Details

Has ligand of interestN

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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: HIV-1 reverse transcriptase initiation complex core / Type: COMPLEX
Details: Peripheral tRNA and engineered stem loop were masked out during cryo-EM processing.
Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.138 MDa / Experimental value: NO
Source (natural)Organism: Human immunodeficiency virus 1
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 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 FIELDBright-field microscopy
Image recordingElectron dose: 100 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.1_3865refinement
PHENIX1.18.1_3865refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
9PHENIXmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1344402 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 64.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00218671
ELECTRON MICROSCOPYf_angle_d0.466512024
ELECTRON MICROSCOPYf_chiral_restr0.03971376
ELECTRON MICROSCOPYf_plane_restr0.00331351
ELECTRON MICROSCOPYf_dihedral_angle_d11.49841530

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