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- EMDB-22900: Structure of HIV-1 reverse transcriptase initiation complex core ... -

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Basic information

Entry
Database: EMDB / ID: EMD-22900
TitleStructure of HIV-1 reverse transcriptase initiation complex core with efavirenz
Map dataStructure of HIV-1 reverse transcriptase initiation complex core with efavirenz
Sample
  • Complex: HIV-1 reverse transcriptase initiation complex core with efavirenz
    • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Protein or peptide: reverse transcriptase p51 subunit
    • RNA: HIV-1 viral RNA fragment
    • Other: tRNA lysine 3 fragment with GNRA tetraloop
  • Ligand: MAGNESIUM ION
  • Ligand: (-)-6-CHLORO-4-CYCLOPROPYLETHYNYL-4-TRIFLUOROMETHYL-1,4-DIHYDRO-2H-3,1-BENZOXAZIN-2-ONE
Function / homology
Function and homology information


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 ...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 / viral penetration into host nucleus / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / lipid binding / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / proteolysis / DNA binding / RNA 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. / Ribonuclease H superfamily / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / 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 / Human immunodeficiency virus type 1 group M subtype B (isolate BH10) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHa B / Larsen KP / Zhang J / Fu Z / Montabana E / Jackson LN / Chen DH / Puglisi EV
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI150464 United States
CitationJournal: Nat Commun / Year: 2021
Title: High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs.
Authors: Betty Ha / Kevin P Larsen / Jingji Zhang / Ziao Fu / Elizabeth Montabana / Lynnette N Jackson / Dong-Hua Chen / Elisabetta Viani Puglisi /
Abstract: Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNA primer bound to the ...Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNA primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNA initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.
History
DepositionOct 26, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMay 12, 2021-
Current statusMay 12, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kjw
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22900.png

Downloads & links

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Map

FileDownload / File: emd_22900.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of HIV-1 reverse transcriptase initiation complex core with efavirenz
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.5
Minimum - Maximum-2.6752973 - 3.8585997
Average (Standard dev.)0.00032532553 (±0.07926496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z314.880314.880314.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ496496496
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-2.6753.8590.000

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Supplemental data

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Sample components

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Entire : HIV-1 reverse transcriptase initiation complex core with efavirenz

EntireName: HIV-1 reverse transcriptase initiation complex core with efavirenz
Components
  • Complex: HIV-1 reverse transcriptase initiation complex core with efavirenz
    • Protein or peptide: Reverse transcriptase/ribonuclease H
    • Protein or peptide: reverse transcriptase p51 subunit
    • RNA: HIV-1 viral RNA fragment
    • Other: tRNA lysine 3 fragment with GNRA tetraloop
  • Ligand: MAGNESIUM ION
  • Ligand: (-)-6-CHLORO-4-CYCLOPROPYLETHYNYL-4-TRIFLUOROMETHYL-1,4-DIHYDRO-2H-3,1-BENZOXAZIN-2-ONE

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Supramolecule #1: HIV-1 reverse transcriptase initiation complex core with efavirenz

SupramoleculeName: HIV-1 reverse transcriptase initiation complex core with efavirenz
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Peripheral tRNA and engineered stem loop were masked out during cryo-EM data processing.
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 138 KDa

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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 group M subtype B (isolate BH10)
Strain: isolate BH10
Molecular weightTheoretical: 64.705316 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 IL

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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
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: isolate BH10
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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Macromolecule #3: HIV-1 viral RNA fragment

MacromoleculeName: HIV-1 viral RNA fragment / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 8.437105 KDa
SequenceString:
GCAGUGGCGC CCGAACAGGG ACUUGA

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Macromolecule #4: tRNA lysine 3 fragment with GNRA tetraloop

MacromoleculeName: tRNA lysine 3 fragment with GNRA tetraloop / type: other / ID: 4 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.595636 KDa
SequenceString:
GCCCGGGAAA CCAGGGUUCA AGUCCCUGUU CGG(G47)CGCCA

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: (-)-6-CHLORO-4-CYCLOPROPYLETHYNYL-4-TRIFLUOROMETHYL-1,4-DIHYDRO-2...

MacromoleculeName: (-)-6-CHLORO-4-CYCLOPROPYLETHYNYL-4-TRIFLUOROMETHYL-1,4-DIHYDRO-2H-3,1-BENZOXAZIN-2-ONE
type: ligand / ID: 6 / Number of copies: 1 / Formula: EFZ
Molecular weightTheoretical: 315.675 Da
Chemical component information

ChemComp-EFZ:
(-)-6-CHLORO-4-CYCLOPROPYLETHYNYL-4-TRIFLUOROMETHYL-1,4-DIHYDRO-2H-3,1-BENZOXAZIN-2-ONE / medication, antiretroviral*YM / Efavirenz

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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

Concentration10.0 mg/mL
BufferpH: 8
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K
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) / Average electron dose: 100.0 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
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 1123557

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7kjw:
Structure of HIV-1 reverse transcriptase initiation complex core with efavirenz

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