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- EMDB-7031: Architecture of HIV-1 reverse transcriptase initiation complex core -

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Entry
Database: EMDB / ID: EMD-7031
TitleArchitecture of HIV-1 reverse transcriptase initiation complex core
Map dataHIV-1 RTIC core
Sample
  • Complex: HIV-1 reverse transcription initiation complex core
    • Complex: HIV-1 reverse transcriptase
      • Protein or peptide: reverse transcriptase p66 subunit
      • Protein or peptide: reverse transcriptase p51 subunit
    • Complex: HIV-1 RNA genome fragment
      • RNA: RNA genome fragment
    • Complex: tRNA lysine3 primer
      • Other: tRNA lysine3
KeywordsReverse 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 / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus ...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 stem-loop binding / RNA-directed DNA polymerase activity / host cell / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / symbiont-mediated suppression of host gene expression / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / lipid binding / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / proteolysis / DNA 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. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLarsen KP / Mathiharan YK / Chen DH / Puglisi JD / Skiniotis G / 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: 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, 2017-
Header (metadata) releaseOct 11, 2017-
Map releaseApr 25, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b19
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7031.png

Downloads & links

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Map

FileDownload / File: emd_7031.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHIV-1 RTIC core
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 224 pix.
= 224. Å		1 Å/pix.
x 224 pix.
= 224. Å		1 Å/pix.
x 224 pix.
= 224. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.06551389 - 0.16009589
Average (Standard dev.)0.00055168907 (±0.0064721378)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 224.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z224.000224.000224.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0660.1600.001

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Supplemental data

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Sample components

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Entire : HIV-1 reverse transcription initiation complex core

EntireName: HIV-1 reverse transcription initiation complex core
Components
  • Complex: HIV-1 reverse transcription initiation complex core
    • Complex: HIV-1 reverse transcriptase
      • Protein or peptide: reverse transcriptase p66 subunit
      • Protein or peptide: reverse transcriptase p51 subunit
    • Complex: HIV-1 RNA genome fragment
      • RNA: RNA genome fragment
    • Complex: tRNA lysine3 primer
      • Other: tRNA lysine3

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Supramolecule #1: HIV-1 reverse transcription initiation complex core

SupramoleculeName: HIV-1 reverse transcription initiation complex core / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Peripheral dynamic RNA elements belonging to tRNA primer and viral RNA template have been masked out for higher resolution structure determination.
Molecular weightTheoretical: 175 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 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 ...Details: The 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.
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 117 KDa

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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: HIV-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.
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 33 KDa

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Supramolecule #4: tRNA lysine3 primer

SupramoleculeName: tRNA lysine3 primer / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Details: Chemically 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 ...Details: Chemically 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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25 KDa

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Macromolecule #1: reverse transcriptase p66 subunit

MacromoleculeName: reverse transcriptase p66 subunit / type: protein_or_peptide / ID: 1 / 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

UniProtKB: Gag-Pol polyprotein

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Macromolecule #2: reverse transcriptase p51 subunit

MacromoleculeName: reverse transcriptase p51 subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
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

UniProtKB: Gag-Pol polyprotein

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Macromolecule #3: RNA genome fragment

MacromoleculeName: RNA genome fragment / type: rna / ID: 3 / 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

GENBANK: GENBANK: AY835748.1

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Macromolecule #4: tRNA lysine3

MacromoleculeName: tRNA lysine3 / type: other / ID: 4 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.720664 KDa
SequenceString:
GCCCGGAUAG CUCAGUCGGU AGAGCAUCAG ACUUUUAAUC UGAGGGUCCA GGGUUCAAGU CCCUGUUCGG (DG)CGCCA (DC)

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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
300.0 mMNaClsodium chloride
10.0 mMC4H11NO3Tris-HCl
0.25 % w/vbeta-octyl glucoside

Details: Beta-OG was added just prior to freezing.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 292 K / Instrument: FEI VITROBOT MARK IV
Details: Blotted for 3.5 sec before plunging into liquid ethane..
DetailsSample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number real images: 4209 / Average exposure time: 8.0 sec. / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 765688
Startup modelType of model: INSILICO MODEL
In silico model: An initial 3D model was obtained using VIPER based on initial selected 2D classes.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 128153
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION

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Atomic model buiding 1

DetailsMain chain backbone for protein
RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

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

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