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- EMDB-21585: +3 extended HIV-1 reverse transcriptase initiation complex core (... -

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Basic information

Entry
Database: EMDB / ID: EMD-21585
Title+3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)
Map data+3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)
Sample
  • Complex: +3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
      • RNA: HIV-1 viral RNA genome fragment
      • Other: tRNA lysine 3
    • Complex: HIV-1 RNA genome fragment
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: tRNA lysine 3
      • Protein or peptide: reverse transcriptase p51 subunit
Function / homology
Function and homology information


integrase activity / viral genome packaging / viral life cycle / Autointegration results in viral DNA circles / Integration of viral DNA into host genomic DNA / 2-LTR circle formation / Vpr-mediated nuclear import of PICs / Plus-strand DNA synthesis / Minus-strand DNA synthesis / Uncoating of the HIV Virion ...integrase activity / viral genome packaging / viral life cycle / Autointegration results in viral DNA circles / Integration of viral DNA into host genomic DNA / 2-LTR circle formation / Vpr-mediated nuclear import of PICs / Plus-strand DNA synthesis / Minus-strand DNA synthesis / Uncoating of the HIV Virion / Early Phase of HIV Life Cycle / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / Binding and entry of HIV virion / Assembly Of The HIV Virion / Budding and maturation of HIV virion / induction by virus of host cysteine-type endopeptidase activity involved in apoptotic process / 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 / peptidase activity / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / lipid binding / DNA-directed DNA polymerase activity / suppression by virus of host gene expression / aspartic-type endopeptidase activity / viral entry into host cell / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / DNA binding / zinc ion binding / membrane / identical protein binding
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 DNA binding domain profile. / Integrase, C-terminal, retroviral / gag gene protein p17 (matrix protein) / Immunodeficiency lentiviral matrix, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Ribonuclease H domain / Retropepsin-like catalytic domain / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Retrovirus capsid, C-terminal / RNase H type-1 domain profile. / Retroviral matrix protein / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Zinc knuckle / Retrovirus capsid, N-terminal / zinc finger / Zinc finger, CCHC-type superfamily / Zinc finger CCHC-type profile. / Zinc finger, CCHC-type / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Gag-Pol polyprotein / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLarsen KP / Jackson LN / Kappel K / Zhang J / Chen DH / 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
DepositionMar 26, 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: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wb2
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21585.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 core (displaced state)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å
1.06 Å/pix.
x 256 pix.
= 271.36 Å
1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.028
Minimum - Maximum-0.03823795 - 0.1018128
Average (Standard dev.)0.00013512948 (±0.003119975)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0380.1020.000

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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 core (...

EntireName: +3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)
Components
  • Complex: +3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
      • RNA: HIV-1 viral RNA genome fragment
      • Other: tRNA lysine 3
    • Complex: HIV-1 RNA genome fragment
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: tRNA lysine 3
      • Protein or peptide: reverse transcriptase p51 subunit

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

SupramoleculeName: +3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Peripheral 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.
Source (natural)Organism: Human immunodeficiency virus 1
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

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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)

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Macromolecule #1: HIV-1 viral RNA genome fragment

MacromoleculeName: HIV-1 viral RNA genome fragment / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 32.623477 KDa
SequenceString:
GACUCUGGUA ACUAGAGAUC CCUCAGACCC UUUUAGUCAG UGUGGAAAAU CUCUAGCAGU GGCGCCCGAA CAGGGACUUG AAAGCGAAA GUAAAGCCAG AG

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Macromolecule #2: tRNA lysine 3

MacromoleculeName: tRNA lysine 3 / type: other / ID: 2 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.354061 KDa
SequenceString:
GCCCGGAUAG CUCAGUCGGU AGAGCAUCAG ACUUUUAAUC UGAGGGUCCA GGGUUCAAGU CCCUGUUCGG GC(DG)CCA (DC)(DT)(DG)

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Macromolecule #3: Reverse transcriptase/ribonuclease H

MacromoleculeName: Reverse transcriptase/ribonuclease H / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 65.55832 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ...String:
MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ILEPFKKQNP DIVIYQYMDD LYVGSDLEIG QHRTKIEELR QHLLRWGLTT PDKKHQKEPP FLWMGYELHP DK WTVQPIV LPEKDSWTVN DICKLVGKLN WASQIYPGIK VRQLSKLLRG TKALTEVIPL TEEAELELAE NREILKEPVH GVY YDPSKD LIAEIQKQGQ GQWTYQIYQE PFKNLKTGKY ARMRGAHTND VKQLTEAVQK ITTESIVIWG KTPKFKLPIQ KETW ETWWT EYWQATWIPE WEFVNTPPLV KLWYQLEKEP IVGAETFYVD GAANRETKLG KAGYVTNKGR QKVVPLTNTT NQKTQ LQAI YLALQDSGLE VNIVTDSQYA LGIIQAQPDK SESELVNQII EQLIKKEKVY LAWVPAHKGI GGNEQVDKLV SAGIRK ILD LGTLVPR

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Macromolecule #4: reverse transcriptase p51 subunit

MacromoleculeName: reverse transcriptase p51 subunit / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 51.585293 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ...String:
MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ILEPFKKQNP DIVIYQYMDD LYVGSDLEIG QHRTKIEELR QHLLRWGLTT PDKKHQKEPP FLWMGYELHP DK WTVQPIV LPEKDSWTVN DIQKLVGKLN WASQIYPGIK VRQLSKLLRG TKALTEVIPL TEEAELELAE NREILKEPVH GVY YDPSKD LIAEIQKQGQ GQWTYQIYQE PFKNLKTGKY ARMRGAHTND VKQLTEAVQK ITTESIVIWG KTPKFKLPIQ KETW ETWWT EYWQATWIPE WEFVNTPPLV KLWYQLEKEP IVGAETF

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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: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 291904

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6wb2:
+3 extended HIV-1 reverse transcriptase initiation complex core (displaced state)

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