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- EMDB-7032: Global architecture of the HIV-1 reverse transcriptase initiation... -

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Basic information

Entry
Database: EMDB / ID: EMD-7032
TitleGlobal architecture of the HIV-1 reverse transcriptase initiation complex
Map dataRTIC global architecture
Sample
  • Complex: HIV-1 reverse transcriptase initiation complex
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
      • Protein or peptide: HIV-1 reverse transcriptase: p66 subunit
      • Protein or peptide: HIV-1 reverse transcriptase: p51 subunit
    • Complex: tRNA lysine3 primer
      • RNA: tRNA Lysine3
    • Complex: HIV-1 RNA genome fragment
      • RNA: HIV-1 viral RNA genome fragment
Biological speciesHuman immunodeficiency virus 1 / Human (human) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 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 releaseMay 2, 2018-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7032.png

Downloads & links

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Map

FileDownload / File: emd_7032.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRTIC global architecture
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.009056951 - 0.08396933
Average (Standard dev.)0.00022913322 (±0.0035869933)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 280.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z280.000280.000280.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0090.0840.000

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Supplemental data

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Sample components

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Entire : HIV-1 reverse transcriptase initiation complex

EntireName: HIV-1 reverse transcriptase initiation complex
Components
  • Complex: HIV-1 reverse transcriptase initiation complex
    • Complex: HIV-1 reverse transcriptaseReverse transcriptase
      • Protein or peptide: HIV-1 reverse transcriptase: p66 subunit
      • Protein or peptide: HIV-1 reverse transcriptase: p51 subunit
    • Complex: tRNA lysine3 primer
      • RNA: tRNA Lysine3
    • Complex: HIV-1 RNA genome fragment
      • RNA: HIV-1 viral RNA genome fragment

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Supramolecule #1: HIV-1 reverse transcriptase initiation complex

SupramoleculeName: HIV-1 reverse transcriptase initiation complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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: 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 ...Details: 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
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 117 KDa

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Supramolecule #3: tRNA lysine3 primer

SupramoleculeName: tRNA lysine3 primer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: Chemically synthesized and extended tRNA lysine3 primer. Modified nucleotide containing a N2-cystamine was placed at position 71. The tRNA primer has been extended by one ddCTP, bringing its ...Details: Chemically synthesized and extended tRNA lysine3 primer. 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.
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 25 KDa

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Supramolecule #4: HIV-1 RNA genome fragment

SupramoleculeName: HIV-1 RNA genome fragment / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Details: HIV-1 RNA genome fragment of 101 nucleotides in length. 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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Macromolecule #1: HIV-1 reverse transcriptase: p66 subunit

MacromoleculeName: HIV-1 reverse transcriptase: p66 subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDFW EVQLGIPHPA GLKKKKSVTV LDVGDAYFSV PLDEDFRKYT AFTIPSINNE TPGIRYQYNV LPQGWKGSPA IFQSSMTKIL ...String:
MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDFW EVQLGIPHPA GLKKKKSVTV LDVGDAYFSV PLDEDFRKYT AFTIPSINNE TPGIRYQYNV LPQGWKGSPA IFQSSMTKIL EPFKKQNPDI VIYQYMDDLY VGSDLEIGQH RTKIEELRQH LLRWGLTTPD KKHQKEPPFL WMGYELHPDK WTVQPIVLPE KDSWTVNDIC KLVGKLNWAS QIYPGIKVRQ LSKLLRGTKA LTEVIPLTEE AELELAENRE ILKEPVHGVY YDPSKDLIAE IQKQGQGQWT YQIYQEPFKN LKTGKYARMR GAHTNDVKQL TEAVQKITTE SIVIWGKTPK FKLPIQKETW ETWWTEYWQA TWIPEWEFVN TPPLVKLWYQ LEKEPIVGAE TFYVDGAANR ETKLGKAGYV TNKGRQKVVP LTNTTNQKTQ LQAIYLALQD SGLEVNIVTD SQYALGIIQA QPDKSESELV NQIIEQLIKK EKVYLAWVPA HKGIGGNEQV DKLVSAGIRK IL

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Macromolecule #2: HIV-1 reverse transcriptase: p51 subunit

MacromoleculeName: HIV-1 reverse transcriptase: p51 subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDFW EVQLGIPHPA GLKKKKSVTV LDVGDAYFSV PLDEDFRKYT AFTIPSINNE TPGIRYQYNV LPQGWKGSPA IFQSSMTKIL ...String:
MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDFW EVQLGIPHPA GLKKKKSVTV LDVGDAYFSV PLDEDFRKYT AFTIPSINNE TPGIRYQYNV LPQGWKGSPA IFQSSMTKIL EPFKKQNPDI VIYQYMDDLY VGSDLEIGQH RTKIEELRQH LLRWGLTTPD KKHQKEPPFL WMGYELHPDK WTVQPIVLPE KDSWTVNDIQ KLVGKLNWAS QIYPGIKVRQ LSKLLRGTKA LTEVIPLTEE AELELAENRE ILKEPVHGVY YDPSKDLIAE IQKQGQGQWT YQIYQEPFKN LKTGKYARMR GAHTNDVKQL TEAVQKITTE SIVIWGKTPK FKLPIQKETW ETWWTEYWQA TWIPEWEFVN TPPLVKLWYQ LEKEPIVGAE TF

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Macromolecule #3: tRNA Lysine3

MacromoleculeName: tRNA Lysine3 / type: rna / ID: 3
Details: 77C is ddC (from RT extension); 71G is dG with an N2-cystamine for cross linking to RT-p66
Source (natural)Organism: Homo sapiens (human)
SequenceString:
GCCCGGAUAG CUCAGUCGGU AGAGCAUCAG ACUUUUAAUC UGAGGGUCCA GGGUUCAAGU CCCUGUUCGG GCGCCAC

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Macromolecule #4: HIV-1 viral RNA genome fragment

MacromoleculeName: HIV-1 viral RNA genome fragment / type: rna / ID: 4
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: NL4.3
SequenceString:
GACUCUGGUA ACUAGAGAUC CCUCAGACCC UUUUAGUCAG UGUGGAAAAU CUCUAGCAGU GGCGCCCGAA CAGGGACUUG AAAGCGAAAG UAAAGCCAGA G

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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 chloridesodium chloride
10.0 mMC4H11NO3Tris-HClTris
0.25 % w/vBOGbeta-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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 765688
CTF correctionSoftware - Name: CTFFIND4
Startup modelType of model: INSILICO MODEL
In silico model: An initial 3D model was obtained using VIPER based on initial selected 2D classes.
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 21520

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