|Entry||Database: PDB / ID: 5u1c|
|Title||Structure of tetrameric HIV-1 Strand Transfer Complex Intasome|
|Descriptor||DNA-binding protein 7d,Integrase/DNA Complex|
|Keywords||VIRAL PROTEIN / integrase / integration / transposase / transesterification|
|Specimen source||Sulfolobus 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
|Method||Electron microscopy (3.9 Å resolution / Particle / Single particle)|
|Authors||Lyumkis, D. / Passos, D.|
|Citation||Science, 2017, 355, 89-92|
Science, 2017, 355, 89-92 Yorodumi Papers
SummaryFull reportAbout validation report
|Date||Deposition: Nov 28, 2016 / Release: Jan 11, 2017|
Downloads & links
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)
-Polypeptide(L) , 1 types, 4 molecules A
B C D
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
-DNA chain , 3 types, 6 molecules G
H E I F J
|#2: DNA chain|
Mass: 3349.197 Da / Num. of mol.: 2 / Source: (synth.) Homo sapiens
|#3: DNA chain|
Mass: 7015.546 Da / Num. of mol.: 2 / Source: (synth.) Human immunodeficiency virus 1
|#4: DNA chain|
Mass: 11414.358 Da / Num. of mol.: 2
Source: (synth.) Human immunodeficiency virus 1, (synth.) Homo sapiens
-Non-polymers , 2 types, 4 molecules
|#5: Chemical||#6: Chemical|
|Sequence details||The protein is a chimera of Sso7d linked to the N-terminus of integrase via an 11-glycine linker.|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE|
|Component||Name: complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer|
Type: COMPLEX / Entity ID: 1,2,3,4 / Source: RECOMBINANT
|Molecular weight||Value: 0.228 deg. / Units: MEGADALTONS / Experimental value: YES|
|Source (natural)||Organism: Human immunodeficiency virus 1|
|Source (recombinant)||Organism: Escherichia coli / Plasmid: pSca355|
|Buffer solution||pH: 8|
|Specimen||Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: GOLD / Grid mesh size: 400 / Grid type: Quantifoil|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 277 kelvins|
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.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELD / Nominal magnification: 22500 / Calibrated magnification: 38167 / Calibrated defocus min: 1500 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 mm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 kelvins / Temperature (min): 90 kelvins|
|Image recording||Average exposure time: 20 sec. / Electron dose: 95 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.
Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 1225
|Image scans||Sampling size: 5 microns / Dimension width: 3838 / Dimension height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100|
|Software||Name: PHENIX / Version: dev_2499: / Classification: refinement|
|CTF correction||Details: performed internally in Relion and Frealign / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Number of particles selected: 274764|
|Symmetry||Point symmetry: C2|
|3D reconstruction||Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 83766 / Algorithm: FOURIER SPACE / Details: Resolution-limited refinement used throughout / Symmetry type: POINT|
|Atomic model building||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.|
Overall b value: 180 / Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: FSC 0.5
-Oct 4, 2017. Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
Three pioneers of this field were awarded Nobel Prize in Chemistry 2017
- Jacques Dubochet (University of Lausanne, Switzerland) is a pioneer of ice-embedding method of EM specimen (as known as cryo-EM), Most of 3DEM structures in EMDB and PDB are obtained using his method.
- Joachim Frank (Columbia University, New York, USA) is a pioneer of single particle reconstruction, which is the most used reconstruction method for 3DEM structures in EMDB and EM entries in PDB. And also, he is a develper of Spider, which is one of the most famous software in this field, and is used for some EM Navigor data (e.g. map projection/slice images).
- Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) was determined the first biomolecule structure by EM. The first EM entry in PDB, PDB-1brd is determinedby him.
External links: The 2017 Nobel Prize in Chemistry - Press Release
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