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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
DescriptorDNA-binding protein 7d,Integrase/DNA Complex
KeywordsVIRAL PROTEIN / integrase / integration / transposase / transesterification
Specimen sourceSulfolobus solfataricus / archaea / thermophilic / スルホロブス・ソルファタリカス
Human immunodeficiency virus 1 / virus / HIV-1 / ヒト免疫不全ウイルス 1
Homo sapiens / human
MethodElectron microscopy (3.9 A resolution / Single particle)
AuthorsLyumkis, D. / Passos, D.
CitationScience, 2017, 355, 89-92

Science, 2017, 355, 89-92 StrPapers
Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.
Dario Oliveira Passos / Min Li / Renbin Yang / Stephanie V Rebensburg / Rodolfo Ghirlando / Youngmin Jeon / Nikoloz Shkriabai / Mamuka Kvaratskhelia / Robert Craigie / Dmitry Lyumkis

DateDeposition: Nov 28, 2016 / Release: Jan 11, 2017 / Last modification: Jan 18, 2017

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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
Polyers212,83910
Non-polymers1794
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (A2)26400
ΔGint (kcal/M)-149
Surface area (A2)49720

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Components

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Polypeptide(L) , 1 types, 4 molecules ABCD

#1: Polypeptide(L)
HIV-1 Integrase, Sso7d chimera / 7 kDa DNA-binding protein d / Sso7d


Mass: 42320.273 Da / Num. of mol.: 4 / Mutation: E152Q
Source: (gene. exp.) Sulfolobus solfataricus, (gene. exp.) Human immunodeficiency virus 1
References: UniProt: A0A157T5S7, UniProt: F2WR39

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DNA chain , 3 types, 6 molecules GHEIFJ

#2: DNA chainDNA (11-MER)


Mass: 3349.197 Da / Num. of mol.: 2 / Source: (synth.) Homo sapiens
#3: DNA chainDNA (23-MER)


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


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

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Non-polymers , 2 types, 4 molecules

#5: ChemicalChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn
#6: ChemicalChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / 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 experimentReconstruction method: SINGLE PARTICLE

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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
Specimen supportGrid material: GOLD / Grid mesh size: 400 /inch. / Grid type: Quantifoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 50
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

MicroscopyMicroscope model: 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 / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 90 K
EM image scansSampling size: 5 microns

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Processing

SoftwareName: PHENIX / Version: dev_2499: / Classification: refinement
ComputingStructure refinement: PHENIX (dev_2499: phenix.real_space_refine)
3D reconstructionResolution: 3.9 A / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 83766 / Details: Resolution-limited refinement used throughout
Atomic model buildingDetails: 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.
Overall b value: 180 / Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: FSC 0.5

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