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

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Basic information

Entry
Database: PDB / ID: 5u1c
TitleStructure of tetrameric HIV-1 Strand Transfer Complex Intasome
Components
  • DNA (11-MER)
  • DNA (23-MER)
  • DNA (37-MER)
  • HIV-1 Integrase, Sso7d chimera
KeywordsVIRAL PROTEIN / integrase / integration / transposase / transesterification
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 / DNA integration / host multivesicular body / 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 / DNA integration / host multivesicular body / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / viral penetration into host nucleus / 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 / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / 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
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 / 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 / Retrovirus capsid, C-terminal / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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
DNA / DNA (> 10) / DNA-binding protein / Gag-Pol polyprotein / Gag-Pol polyprotein
Similarity search - Component
Biological speciesSulfolobus solfataricus (archaea)
Human immunodeficiency virus 1
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLyumkis, D. / Passos, D.
Funding support United States, 3items
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2017Group: Database references / Source and taxonomy
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support
Item: _pdbx_audit_support.funding_organization / _pdbx_audit_support.grant_number
Revision 1.4Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: HIV-1 Integrase, Sso7d chimera
B: HIV-1 Integrase, Sso7d chimera
G: DNA (11-MER)
E: DNA (23-MER)
F: DNA (37-MER)
C: HIV-1 Integrase, Sso7d chimera
D: HIV-1 Integrase, Sso7d chimera
H: DNA (11-MER)
I: DNA (23-MER)
J: DNA (37-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,01914
Polymers212,83910
Non-polymers1794
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26400 Å2
ΔGint-149 kcal/mol
Surface area49720 Å2

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
HIV-1 Integrase, Sso7d chimera / 7 kDa DNA-binding protein d / Sso7d


Mass: 42320.273 Da / Num. of mol.: 4 / Mutation: E152Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus solfataricus (archaea), (gene. exp.) Human immunodeficiency virus 1
Gene: SSOP1_2677, pol / Production host: Escherichia coli (E. coli)
References: UniProt: A0A157T5S7, UniProt: F2WR39, UniProt: P12497*PLUS

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DNA chain , 3 types, 6 molecules GHEIFJ

#2: DNA chain DNA (11-MER)


Mass: 3349.197 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (23-MER)


Mass: 7015.546 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1
#4: DNA chain DNA (37-MER)


Mass: 11414.358 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Human immunodeficiency virus 1, (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Sequence detailsThe protein is a chimera of Sso7d linked to the N-terminus of integrase via an 11-glycine linker.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.228 MDa / Experimental value: YES
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pSca355
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1500 mg/mLsodium chlorideNaCl1
220 mMTrisC4H11NO31
30.5 mMTCEPC9H15O6P1
46 %glycerolC3H8O31
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 277 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Calibrated magnification: 38167 X / Calibrated defocus min: 1500 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 90 K
Image recordingAverage exposure time: 20 sec. / Electron dose: 95 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1225
Details: Individual frames were gain-corrected, aligned, and summed with the application of an exposure filter using MotionCor2, according to the nominal dose rate.
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100

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Processing

SoftwareName: PHENIX / Version: dev_2499: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2Leginon3.2image acquisition
4CTFFIND3CTF correction
7Rosetta2015.12.57698_bundlemodel fitting
8PHENIX2499model fitting
9Cootmodel fitting
11RELION1.3initial Euler assignment
12FREALIGN9.11final Euler assignment
13FREALIGN3.11classification
14FREALIGN9.113D reconstruction
15PHENIX2499model refinementreal space refinement
16Cootmodel refinementfinal details fixed and fitted manually
CTF correctionDetails: performed internally in Relion and Frealign / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 274764
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83766 / Algorithm: FOURIER SPACE / Details: Resolution-limited refinement used throughout / Symmetry type: POINT
Atomic model buildingB value: 180 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: FSC 0.5
Details: To 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) ...Details: To 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.

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