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- EMDB-22004: +3 extended HIV-1 reverse transcriptase initiation complex (inter... -

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Basic information

Entry
Database: EMDB / ID: EMD-22004
Title+3 extended HIV-1 reverse transcriptase initiation complex (intermediate state, unmasked map)
Map data+3 extended HIV-1 reverse transcriptase initiation complex
Sample
  • Complex: +3 extended HIV-1 reverse transcriptase initiation complex (intermediate state)
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
    • Complex: HIV-1 RNA genome fragment
    • Complex: tRNA lysine 3
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsLarsen KP / Jackson LN / Zhang J / Puglisi EV
Funding support United States, 1 items
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
DepositionMay 20, 2020-
Header (metadata) releaseJun 24, 2020-
Map releaseJun 24, 2020-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22004.png

Downloads & links

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Map

FileDownload / File: emd_22004.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation+3 extended HIV-1 reverse transcriptase initiation complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.011375891 - 0.0559148
Average (Standard dev.)0.00013979654 (±0.0025255256)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0110.0560.000

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Supplemental data

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Sample components

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Entire : +3 extended HIV-1 reverse transcriptase initiation complex (inter...

EntireName: +3 extended HIV-1 reverse transcriptase initiation complex (intermediate state)
Components
  • Complex: +3 extended HIV-1 reverse transcriptase initiation complex (intermediate state)
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
    • Complex: HIV-1 RNA genome fragment
    • Complex: tRNA lysine 3

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Supramolecule #1: +3 extended HIV-1 reverse transcriptase initiation complex (inter...

SupramoleculeName: +3 extended HIV-1 reverse transcriptase initiation complex (intermediate state)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25 KDa

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Supramolecule #2: HIV-1 reverse transcriptase

SupramoleculeName: HIV-1 reverse transcriptase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Details: The C-terminus of p66 contains an unstructured linker and a six-histidine tag that was cleaved prior to full complex formation.
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: HIV-1 RNA genome fragment

SupramoleculeName: HIV-1 RNA genome fragment / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: A fragment of the HIV-1 RNA genome that is 101 nucleotides in length.
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: tRNA lysine 3

SupramoleculeName: tRNA lysine 3 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Details: Chemically 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 ...Details: Chemically 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.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration5.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
75.0 mMNaClSodium chloridesodium chloride
10.0 mMC4H11NO3tris
6.0 mMMgCl2magnesium chloride
0.2 % w/vbeta-octyl glucoside

Details: Full complex was prepared 5-8 hours before freezing. Beta-OG was added just prior to freezing.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 292 K / Instrument: LEICA EM GP / Details: 3 microliters applied, 2s pre-blot, 2.5s blot..
DetailsSample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 77.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 157727

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