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- EMDB-8481: Structure of tetrameric HIV-1 Strand Transfer Complex Intasome -

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Entry
Database: EMDB / ID: EMD-8481
TitleStructure of tetrameric HIV-1 Strand Transfer Complex Intasome
Map datacryoEM reconstruction of a tetrameric HIV-1 strand transfer complex intasome
Sample
  • Complex: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer
    • Protein or peptide: HIV-1 Integrase, Sso7d chimera
    • DNA: DNA (11-MER)
    • DNA: DNA (23-MER)
    • DNA: DNA (37-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
Keywordsintegrase / integration / transposase / transesterification / VIRAL PROTEIN
Function / homology
Function and homology information


RNA endonuclease 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 endonuclease 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 / symbiont-mediated suppression of host gene expression / 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 / endonuclease activity / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / 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
DNA-binding 7kDa protein / 7kD DNA-binding domain / Chromo-like domain superfamily / 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 ...DNA-binding 7kDa protein / 7kD DNA-binding domain / Chromo-like domain superfamily / 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 / RNase H type-1 domain profile. / Ribonuclease H domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase (RNA-dependent DNA polymerase) / Retroviral matrix protein / Retrovirus capsid, C-terminal / 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. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA-binding protein / Integrase / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLyumkis D / Passos D
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103368 United States
Leona M. and Harry B. Helmsley Charitble Trust Grant#2012-PG-MED002 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)Intramural Program of the National Institute of Diabetes and Digestive Diseases United States
CitationJournal: Science / Year: 2017
Title: Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.
Authors: Dario Oliveira Passos / Min Li / Renbin Yang / Stephanie V Rebensburg / Rodolfo Ghirlando / Youngmin Jeon / Nikoloz Shkriabai / Mamuka Kvaratskhelia / Robert Craigie / Dmitry Lyumkis /
Abstract: Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase ...Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo-electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements. The distinct STC structures highlight how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for assembly.
History
DepositionNov 28, 2016-
Header (metadata) releaseJan 11, 2017-
Map releaseJan 11, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5u1c
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8481.png

Downloads & links

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Map

FileDownload / File: emd_8481.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM reconstruction of a tetrameric HIV-1 strand transfer complex intasome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 192 pix.
= 251.52 Å		1.31 Å/pix.
x 192 pix.
= 251.52 Å		1.31 Å/pix.
x 192 pix.
= 251.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.188862 - 0.36068627
Average (Standard dev.)0.00052015175 (±0.011994169)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 251.51999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z251.520251.520251.520
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.1890.3610.001

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Supplemental data

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Half map: cryoEM reconstruction of a tetrameric HIV-1 strand transfer...

Fileemd_8481_half_map_1.map
AnnotationcryoEM reconstruction of a tetrameric HIV-1 strand transfer complex intasome, map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryoEM reconstruction of a tetrameric HIV-1 strand transfer...

Fileemd_8481_half_map_2.map
AnnotationcryoEM reconstruction of a tetrameric HIV-1 strand transfer complex intasome, map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Int...

EntireName: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer
Components
  • Complex: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer
    • Protein or peptide: HIV-1 Integrase, Sso7d chimera
    • DNA: DNA (11-MER)
    • DNA: DNA (23-MER)
    • DNA: DNA (37-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Int...

SupramoleculeName: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 228 KDa

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Macromolecule #1: HIV-1 Integrase, Sso7d chimera

MacromoleculeName: HIV-1 Integrase, Sso7d chimera / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 42.320273 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKGGGGG GGGGGGFLDG IDKAQEEHEK YHSNWRAMAS DFNLPPVVAK EIVASCDKCQ LKGEAMHGQV DCSPGIWQLD C THLEGKVI ...String:
MGSSHHHHHH SSGLVPRGSH MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKGGGGG GGGGGGFLDG IDKAQEEHEK YHSNWRAMAS DFNLPPVVAK EIVASCDKCQ LKGEAMHGQV DCSPGIWQLD C THLEGKVI LVAVHVASGY IEAEVIPAET GQETAYFLLK LAGRWPVKTV HTDNGSNFTS TTVKAACWWA GIKQEFGIPY NP QSQGVIQ SMNKELKKII GQVRDQAEHL KTAVQMAVFI HNFKRKGGIG GYSAGERIVD IIATDIQTKE LQKQITKIQN FRV YYRDSR DPVWKGPAKL LWKGEGAVVI QDNSDIKVVP RRKAKIIRDY GKQMAGDDCV ASRQDED

UniProtKB: DNA-binding protein, Integrase

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Macromolecule #2: DNA (11-MER)

MacromoleculeName: DNA (11-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.349197 KDa
SequenceString:
(DG)(DT)(DA)(DC)(DG)(DC)(DT)(DG)(DA)(DC) (DT)

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Macromolecule #3: DNA (23-MER)

MacromoleculeName: DNA (23-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 7.015546 KDa
SequenceString:
(DA)(DC)(DT)(DG)(DC)(DT)(DA)(DG)(DA)(DG) (DA)(DT)(DT)(DT)(DT)(DC)(DC)(DA)(DC)(DA) (DC)(DT)(DG)

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Macromolecule #4: DNA (37-MER)

MacromoleculeName: DNA (37-MER) / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.414358 KDa
SequenceString:
(DC)(DA)(DG)(DT)(DG)(DT)(DG)(DG)(DA)(DA) (DA)(DA)(DT)(DC)(DT)(DC)(DT)(DA)(DG)(DC) (DA)(DG)(DT)(DT)(DA)(DC)(DA)(DG)(DT) (DC)(DA)(DG)(DC)(DG)(DT)(DA)(DC)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
500.0 mg/mLNaClsodium chloride
20.0 mMC4H11NO3Tris
0.5 mMC9H15O6PTCEP
6.0 %C3H8O3glycerol
GridModel: Quantifoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 50 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Sample containing HIV STC intasomes in SEC buffer was applied onto freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey gold UltrAuFoil grids (Quantifoil), adsorbed for 30 ...Details: Sample containing HIV STC intasomes in SEC buffer was applied onto freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey gold UltrAuFoil grids (Quantifoil), adsorbed for 30 seconds, then plunged into liquid ethane using a manual cryo-plunger in an ambient environment of 4 degrees C..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 90.0 K / Max: 90.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-100 / Number grids imaged: 1 / Number real images: 1225 / Average exposure time: 20.0 sec. / Average electron dose: 95.0 e/Å2
Details: Individual frames were gain-corrected, aligned, and summed with the application of an exposure filter using MotionCor2, according to the nominal dose rate.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
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

Particle selectionNumber selected: 274764
Startup modelType of model: INSILICO MODEL / In silico model: common lines model using OptiMod
Details: An initial model was generated directly from the class averages using OptiMod.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Details: Resolution-limited refinement used throughout / Number images used: 83766
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 1.3) / Details: Relion 3D classification, auto mode
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11) / Details: Frealign 3D classification and refinement
Final 3D classificationSoftware - Name: FREALIGN (ver. 3.11)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsTo generate ensemble models, the complete intasome model was iteratively relaxed - using two-fold symmetry and a combination of Rosetta and Phenix - against one half map (the working map) and inspected for consistency with the second half map (the free map). The model was then adjusted manually using Coot. Final ensemble modeling used half maps for all aspects of refinement and evaluation: 500 models were generated as described using Rosetta. From the 100 top-scoring models (scored by Rosetta energy), the ten models with the best map-to-model FSC were selected and refined in real space using secondary-structure restraints in Phenix. Molprobity was used throughout the refinement process.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 180 / Target criteria: FSC 0.5
Output model

PDB-5u1c:
Structure of tetrameric HIV-1 Strand Transfer Complex Intasome

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