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- EMDB-43114: Kinetic intermediate states of HIV-1 RT DNA synthesis captured by... -

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Basic information

Entry
Database: EMDB / ID: EMD-43114
TitleKinetic intermediate states of HIV-1 RT DNA synthesis captured by cryo-EM
Map data
Sample
  • Complex: Reverse transcriptase/ribonuclease H in complex with DNA aptamer
    • Protein or peptide: HIV-1 reverse transcriptase/ribonuclease H P66 subunit
    • Protein or peptide: HIV-1 reverse transcriptase P51 subunit
    • DNA: DNA (38-MER)
KeywordsReverse Transcription / Time-resolved / HIV-1 / Cryo-EM / TRANSCRIPTION / TRANSFERASE-DNA complex
Function / homology
Function and homology information


HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / symbiont-mediated activation of host apoptosis / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase ...HIV-1 retropepsin / retroviral ribonuclease H / exoribonuclease H / symbiont-mediated activation of host apoptosis / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase / viral penetration into host nucleus / symbiont-mediated suppression of host gene expression / 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 entry into host cell / viral translational frameshifting / 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. / 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 / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / 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. / 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 1 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsVergara S / Zhou X / Santiago U / Conway JF / Sluis-Cremer N / Calero G
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI175067 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082251 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription.
Authors: Sandra Vergara / Xiaohong Zhou / Ulises Santiago / Mounia Alaoui-El-Azher / James F Conway / Nicolas Sluis-Cremer / Guillermo Calero /
Abstract: Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight ...Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg (site B) inducing formation of a negatively charged pocket where a second floating Mg can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg and the primer 3'-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.
History
DepositionDec 12, 2023-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43114.png

Downloads & links

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Map

FileDownload / File: emd_43114.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 316.8 Å		0.83 Å/pix.
x 384 pix.
= 316.8 Å		0.83 Å/pix.
x 384 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.7914169 - 1.6242471
Average (Standard dev.)-0.000077557095 (±0.026015928)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43114_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43114_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43114_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 : Reverse transcriptase/ribonuclease H in complex with DNA aptamer

EntireName: Reverse transcriptase/ribonuclease H in complex with DNA aptamer
Components
  • Complex: Reverse transcriptase/ribonuclease H in complex with DNA aptamer
    • Protein or peptide: HIV-1 reverse transcriptase/ribonuclease H P66 subunit
    • Protein or peptide: HIV-1 reverse transcriptase P51 subunit
    • DNA: DNA (38-MER)

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Supramolecule #1: Reverse transcriptase/ribonuclease H in complex with DNA aptamer

SupramoleculeName: Reverse transcriptase/ribonuclease H in complex with DNA aptamer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Human immunodeficiency virus 1

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Macromolecule #1: HIV-1 reverse transcriptase/ribonuclease H P66 subunit

MacromoleculeName: HIV-1 reverse transcriptase/ribonuclease H P66 subunit
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 64.104457 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 SAG

UniProtKB: Gag-Pol polyprotein

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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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 51.944691 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAHHHHHHAL EVLFQGPISP IETVPVKLKP GMDGPKVKQW PLTEEKIKAL VEICTEMEKE GKISKIGPEN PYNTPVFAIK KKDSTKWRK LVDFRELNKR TQDFWEVQLG IPHPAGLKKK KSVTVLDVGD AYFSVPLDED FRKYTAFTIP SINNETPGIR Y QYNVLPQG ...String:
MAHHHHHHAL EVLFQGPISP IETVPVKLKP GMDGPKVKQW PLTEEKIKAL VEICTEMEKE GKISKIGPEN PYNTPVFAIK KKDSTKWRK LVDFRELNKR TQDFWEVQLG IPHPAGLKKK KSVTVLDVGD AYFSVPLDED FRKYTAFTIP SINNETPGIR Y QYNVLPQG WKGSPAIFQS SMTKILEPFK KQNPDIVIYQ YMDDLYVGSD LEIGQHRTKI EELRQHLLRW GLTTPDKKHQ KE PPFLWMG YELHPDKWTV QPIVLPEKDS WTVNDIQKLV GKLNWASQIY PGIKVRQLCK LLRGTKALTE VIPLTEEAEL ELA ENREIL KEPVHGVYYD PSKDLIAEIQ KQGQGQWTYQ IYQEPFKNLK TGKYARMRGA HTNDVKQLTE AVQKITTESI VIWG KTPKF KLPIQKETWE TWWTEYWQAT WIPEWEFVNT PPLVKLWYQ

UniProtKB: Gag-Pol polyprotein

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Macromolecule #3: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 3 / Details: DNA aptamer / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.739513 KDa
SequenceString:
(DT)(DA)(DA)(DT)(DT)(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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4
GridMaterial: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 96000
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

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5d3g

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 488216
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8vb6:
Kinetic intermediate states of HIV-1 RT DNA synthesis captured by cryo-EM

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