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- EMDB-13156: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptam... -

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Entry
Database: EMDB / ID: EMD-13156
TitleCryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket
Map data
Sample
  • Complex: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: DNA (37-mer)
      • DNA: DNA (37-MER)
  • Ligand: (1~{R},2~{R})-~{N}-(1~{H}-pyrazol-4-yl)-2-pyridin-3-yl-cyclopropane-1-carboxamide
KeywordsReverse transcriptase / RT-aptamer complex / RT sliding / P-1 complex / P51 / P66 / TRANSFERASE
Function / homology
Function and homology information


RNA-directed DNA polymerase activity / HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase ...RNA-directed DNA polymerase activity / HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / 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 / telomerase activity / viral penetration into host nucleus / RNA stem-loop binding / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / host cell / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / symbiont-mediated suppression of host gene expression / symbiont entry into host cell / viral translational frameshifting / lipid binding / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / 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. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 BH10 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsSingh AK / Das K
CitationJournal: Nat Commun / Year: 2021
Title: Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening.
Authors: Abhimanyu K Singh / Sergio E Martinez / Weijie Gu / Hoai Nguyen / Dominique Schols / Piet Herdewijn / Steven De Jonghe / Kalyan Das /
Abstract: HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the ...HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.
History
DepositionJul 1, 2021-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p15
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13156.png

Downloads & links

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Map

FileDownload / File: emd_13156.map.gz / Format: CCP4 / Size: 7.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 125 pix.
= 121.25 Å		0.97 Å/pix.
x 125 pix.
= 121.25 Å		0.97 Å/pix.
x 125 pix.
= 121.25 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0184 / Movie #1: 0.035
Minimum - Maximum-0.25295013 - 0.3458422
Average (Standard dev.)0.0000797285 (±0.018701795)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions125125125
Spacing125125125
CellA=B=C: 121.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.970.970.97
M x/y/z125125125
origin x/y/z0.0000.0000.000
length x/y/z121.250121.250121.250
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS125125125
D min/max/mean-0.2530.3460.000

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Supplemental data

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Half map: #1

Fileemd_13156_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13156_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptam...

EntireName: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket
Components
  • Complex: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: DNA (37-mer)
      • DNA: DNA (37-MER)
  • Ligand: (1~{R},2~{R})-~{N}-(1~{H}-pyrazol-4-yl)-2-pyridin-3-yl-cyclopropane-1-carboxamide

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Supramolecule #1: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptam...

SupramoleculeName: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Human immunodeficiency virus type 1 BH10
Molecular weightTheoretical: 11.43 KDa

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Supramolecule #2: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT

SupramoleculeName: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Human immunodeficiency virus type 1 BH10

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Supramolecule #3: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT

SupramoleculeName: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Human immunodeficiency virus type 1 BH10

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Supramolecule #4: DNA (37-mer)

SupramoleculeName: DNA (37-mer) / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3 / Details: Synthetic DNA construct
Source (natural)Organism: Human immunodeficiency virus type 1 BH10

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Macromolecule #1: Reverse transcriptase/ribonuclease H

MacromoleculeName: Reverse transcriptase/ribonuclease H / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Human immunodeficiency virus type 1 BH10
Molecular weightTheoretical: 64.047398 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 IVGAETFYVD GAANRETKLG KAGYVTNKGR QKVVPLTNTT NQKTE LQAI YLALQDSGLE VNIVTNSQYA LGIIQAQPDK SESELVNQII EQLIKKEKVY LAWVPAHKGI GGNEQVDKLV SA

UniProtKB: Gag-Pol polyprotein

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Macromolecule #2: Reverse transcriptase/ribonuclease H

MacromoleculeName: Reverse transcriptase/ribonuclease H / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Human immunodeficiency virus type 1 BH10
Molecular weightTheoretical: 50.039488 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWE VQLGIPHPAG LKKKKSVTVL DVGDAYFSVP LDEDFRKYTA FTIPSINNET PGIRYQYNVL PQGWKGSPAI F QSSMTKIL ...String:
PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWE VQLGIPHPAG LKKKKSVTVL DVGDAYFSVP LDEDFRKYTA FTIPSINNET PGIRYQYNVL PQGWKGSPAI F QSSMTKIL EPFKKQNPDI VIYQYMDDLY VGSDLEIGQH RTKIEELRQH LLRWGLTTPD KKHQKEPPFL WMGYELHPDK WT VQPIVLP EKDSWTVNDI QKLVGKLNWA SQIYPGIKVR QLSKLLRGTK ALTEVIPLTE EAELELAENR EILKEPVHGV YYD PSKDLI AEIQKQGQGQ WTYQIYQEPF KNLKTGKYAR MRGAHTNDVK QLTEAVQKIT TESIVIWGKT PKFKLPIQKE TWET WWTEY WQATWIPEWE FVNTPPLVKL WYQ

UniProtKB: Gag-Pol polyprotein

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Macromolecule #3: DNA (37-MER)

MacromoleculeName: DNA (37-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.434332 KDa
SequenceString:
(DT)(DA)(DA)(DT)(DA)(DT)(OMC)(DC)(OMC)(DC) (DC)(DC)(DT)(DT)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DT)(DT)(DG)(DC)(DA)(DC)(DC)(DG) (DA)(DA)(DG)(DG)(DG)(DG)(DG)(DG)

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Macromolecule #4: (1~{R},2~{R})-~{N}-(1~{H}-pyrazol-4-yl)-2-pyridin-3-yl-cyclopropa...

MacromoleculeName: (1~{R},2~{R})-~{N}-(1~{H}-pyrazol-4-yl)-2-pyridin-3-yl-cyclopropane-1-carboxamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: 4OI
Molecular weightTheoretical: 228.25 Da
Chemical component information

ChemComp-4OI:
(1~{R},2~{R})-~{N}-(1~{H}-pyrazol-4-yl)-2-pyridin-3-yl-cyclopropane-1-carboxamide

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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

Concentration0.4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMNH2C(CH2OH)3HClTris-HCl
75.0 mMNaClNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 767 / Average exposure time: 55.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 733223
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5hp1

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 157094
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5hp1


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 148.9 / Target criteria: Correlation coefficient
Output model

PDB-7p15:
Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket

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