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- EMDB-8483: Structure of higher-order HIV-1 strand transfer complex intasome -

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Basic information

Entry
Database: EMDB / ID: EMD-8483
TitleStructure of higher-order HIV-1 strand transfer complex intasome
Map datastructure of higher-order HIV-1 Strand Transfer Complex Intasome
Sample
  • Complex: complex formed by a higher-order assembly of Sso7d-fusion HIV-1 Integrase with with IN-binding domain of LEDGF/p75, and the product of DNA strand transfer
    • Protein or peptide: HIV-1 Integrase, Sso7d chimera
  • Protein or peptide: Integrase binding domain of LEDGF/p75
  • DNA: DNA (11-MER)
  • DNA: DNA (23-MER)
  • DNA: DNA (37-MER)
Function / homology
Function and homology information


RNA endonuclease activity / nucleotidyltransferase activity / HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration ...RNA endonuclease activity / nucleotidyltransferase activity / 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 / endonuclease activity / DNA recombination / Hydrolases; Acting on ester bonds / nucleic acid binding / 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
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. / 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
DNA-binding protein / Integrase / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsLyumkis D / Passos D
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-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8483.png

Downloads & links

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Map

FileDownload / File: emd_8483.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of higher-order HIV-1 Strand Transfer Complex Intasome
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.12662712 - 0.25486803
Average (Standard dev.)0.0015244725 (±0.009931429)
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-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.1270.2550.002

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Supplemental data

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Sample components

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Entire : complex formed by a higher-order assembly of Sso7d-fusion HIV-1 I...

EntireName: complex formed by a higher-order assembly of Sso7d-fusion HIV-1 Integrase with with IN-binding domain of LEDGF/p75, and the product of DNA strand transfer
Components
  • Complex: complex formed by a higher-order assembly of Sso7d-fusion HIV-1 Integrase with with IN-binding domain of LEDGF/p75, and the product of DNA strand transfer
    • Protein or peptide: HIV-1 Integrase, Sso7d chimera
  • Protein or peptide: Integrase binding domain of LEDGF/p75
  • DNA: DNA (11-MER)
  • DNA: DNA (23-MER)
  • DNA: DNA (37-MER)

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Supramolecule #1: complex formed by a higher-order assembly of Sso7d-fusion HIV-1 I...

SupramoleculeName: complex formed by a higher-order assembly of Sso7d-fusion HIV-1 Integrase with with IN-binding domain of LEDGF/p75, and the product of DNA strand transfer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pSca355
Molecular weightExperimental: 500 KDa

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Macromolecule #1: HIV-1 Integrase, Sso7d chimera

MacromoleculeName: HIV-1 Integrase, Sso7d chimera / type: protein_or_peptide / ID: 1
Details: Sso7d protein fused to the integrase N-terminus via an 11-glycine linker
Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKGGGGGG GGGGGFLDGI DKAQEEHEKY HSNWRAMASD FNLPPVVAKE IVASCDKCQL KGEAMHGQVD CSPGIWQLDC THLEGKVILV ...String:
MGSSHHHHHH SSGLVPRGSH MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKGGGGGG GGGGGFLDGI DKAQEEHEKY HSNWRAMASD FNLPPVVAKE IVASCDKCQL KGEAMHGQVD CSPGIWQLDC THLEGKVILV AVHVASGYIE AEVIPAETGQ ETAYFLLKLA GRWPVKTVHT DNGSNFTSTT VKAACWWAGI KQEFGIPYNP QSQGVIQSMN KELKKIIGQV RDQAEHLKTA VQMAVFIHNF KRKGGIGGYS AGERIVDIIA TDIQTKELQK QITKIQNFRV YYRDSRDPVW KGPAKLLWKG EGAVVIQDNS DIKVVPRRKA KIIRDYGKQM AGDDCVASRQ DED

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Macromolecule #2: Integrase binding domain of LEDGF/p75

MacromoleculeName: Integrase binding domain of LEDGF/p75 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
SMDSRLQRIH AEIKNSLKID NLDVNRCIEA LDELASLQVT MQQAQKHTEM ITTLKKIRRF KVSQVIMEKS TMLYNKFKNM FLV

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Macromolecule #3: DNA (11-MER)

MacromoleculeName: DNA (11-MER) / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
GTACGCTGAC T

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Macromolecule #4: DNA (23-MER)

MacromoleculeName: DNA (23-MER) / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Human immunodeficiency virus 1
SequenceString:
ACTGCTAGAG ATTTTCCACA CTG

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Macromolecule #5: DNA (37-MER)

MacromoleculeName: DNA (37-MER) / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
CAGTGTGGAA AATCTCTAGC AGTTACAGTC AGCGTAC

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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
650.0 mg/mLNaClSodium chloridesodium chloride
20.0 mMC4H11NO3Tris
0.5 mMC9H15O6PTCEP
6.0 %C3H8O3glycerol
GridModel: Quantifoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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 - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-100 / Number grids imaged: 1 / Number real images: 1598 / 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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 154445
CTF correctionSoftware - Name: CTFFIND (ver. 3) / Details: performed internally in Relion and Frealign
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.
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 3D classificationSoftware - Name: FREALIGN (ver. 3.11)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11) / Details: Frealign 3D classification and refinement
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Details: Resolution-limited refinement used throughout / Number images used: 11099

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT / Overall B value: 200

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