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- EMDB-14458: Cryo-EM structure of NNRTI resistant M184I/E138K mutant HIV-1 rev... -

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Entry
Database: EMDB / ID: EMD-14458
TitleCryo-EM structure of NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase with a DNA aptamer in complex with nevirapine
Map data
Sample
  • Complex: NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase with a DNA aptamer in complex with nevirapine
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT with M184I mutation
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT with E138K mutation
      • Protein or peptide: p51 RT
    • DNA: DNA (38-MER)
  • Ligand: 11-CYCLOPROPYL-5,11-DIHYDRO-4-METHYL-6H-DIPYRIDO[3,2-B:2',3'-E][1,4]DIAZEPIN-6-ONE
KeywordsReverse transcriptase mutant / RT-aptamer complex / NNRTI resistance / Non-nucleoside inhibitor / TRANSFERASE
Function / homology
Function and homology information


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 ...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 / viral penetration into host nucleus / RNA stem-loop binding / RNA-directed DNA polymerase activity / 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 / viral translational frameshifting / lipid binding / symbiont entry into host cell / 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: 3.38 Å
AuthorsSingh AK / Das K
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine.
Authors: Abhimanyu K Singh / Brent De Wijngaert / Marc Bijnens / Kris Uyttersprot / Hoai Nguyen / Sergio E Martinez / Dominique Schols / Piet Herdewijn / Christophe Pannecouque / Eddy Arnold / Kalyan Das /
Abstract: Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles ...Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.
History
DepositionFeb 26, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14458.png

Downloads & links

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Map

FileDownload / File: emd_14458.map.gz / Format: CCP4 / Size: 7.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.31102663 - 0.40840542
Average (Standard dev.)0.00023245197 (±0.02176636)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Half map: #1

Fileemd_14458_half_map_1.map
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Half map: #2

Fileemd_14458_half_map_2.map
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Sample components

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Entire : NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase wi...

EntireName: NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase with a DNA aptamer in complex with nevirapine
Components
  • Complex: NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase with a DNA aptamer in complex with nevirapine
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT with M184I mutation
      • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Complex: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT with E138K mutation
      • Protein or peptide: p51 RT
    • DNA: DNA (38-MER)
  • Ligand: 11-CYCLOPROPYL-5,11-DIHYDRO-4-METHYL-6H-DIPYRIDO[3,2-B:2',3'-E][1,4]DIAZEPIN-6-ONE

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Supramolecule #1: NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase wi...

SupramoleculeName: NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase with a DNA aptamer in complex with nevirapine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Human immunodeficiency virus type 1 BH10

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Supramolecule #2: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT with M184I mutation

SupramoleculeName: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT with M184I mutation
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 with E138K mutation

SupramoleculeName: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT with E138K mutation
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
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 / Details: P66 subunit / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Human immunodeficiency virus type 1 BH10 / Strain: isolate BH10
Molecular weightTheoretical: 64.019352 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFACKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ...String:
MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFACKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ILEPFKKQNP DIVIYQYIDD 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: p51 RT

MacromoleculeName: p51 RT / type: protein_or_peptide / ID: 2 / Details: P51 subunit / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus type 1 BH10 / Strain: isolate BH10
Molecular weightTheoretical: 50.039555 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWE VQLGIPHPAG LKKKKSVTVL DVGDAYFSVP LDEDFRKYTA FTIPSINNKT PGIRYQYNVL PQGWKGSPAI F QSSMTKIL ...String:
PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWE VQLGIPHPAG LKKKKSVTVL DVGDAYFSVP LDEDFRKYTA FTIPSINNKT 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 (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.748526 KDa
SequenceString:
(DT)(DA)(DA)(DT)(DA)(DC)(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)(DG)

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Macromolecule #4: 11-CYCLOPROPYL-5,11-DIHYDRO-4-METHYL-6H-DIPYRIDO[3,2-B:2',3'-E][1...

MacromoleculeName: 11-CYCLOPROPYL-5,11-DIHYDRO-4-METHYL-6H-DIPYRIDO[3,2-B:2',3'-E][1,4]DIAZEPIN-6-ONE
type: ligand / ID: 4 / Number of copies: 1 / Formula: NVP
Molecular weightTheoretical: 266.298 Da
Chemical component information

ChemComp-NVP:
11-CYCLOPROPYL-5,11-DIHYDRO-4-METHYL-6H-DIPYRIDO[3,2-B:2',3'-E][1,4]DIAZEPIN-6-ONE / medication*YM

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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

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMNH2C(CH2OH)3HClTris-HCl
75.0 mMNaClNaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / 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: 1981 / Average exposure time: 55.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.5 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 stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1323229
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5hp1

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 304552
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: 163.4 / Target criteria: Correlation coefficient
Output model

PDB-7z29:
Cryo-EM structure of NNRTI resistant M184I/E138K mutant HIV-1 reverse transcriptase with a DNA aptamer in complex with nevirapine

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