|Entry||Database: EMDB / ID: 8483|
|Title||Structure of higher-order HIV-1 strand transfer complex intasome|
|Sample||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|
|Source||Human immunodeficiency virus 1 / virus / ヒト免疫不全ウイルス 1|
|Map data||structure of higher-order HIV-1 Strand Transfer Complex Intasome|
|Method||single particle reconstruction, at 4.6 A resolution|
|Authors||Lyumkis D / Passos D|
|Citation||Science, 2017, 355, 89-92|
|Date||Deposition: Nov 28, 2016 / Header (metadata) release: Jan 11, 2017 / Map release: Jan 11, 2017 / Last update: Nov 28, 2016|
Downloads & links
|File||emd_8483.map.gz (map file in CCP4 format, 28312 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1.31 A|
CCP4 map header:
+Entire complex formed by a higher-order assembly of Sso7d-fusion HIV-1 I...
|Entire||Name: 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|
Number of components: 6
|Mass||Experimental: 500 kDa|
+Component #1: protein, complex formed by a higher-order assembly of Sso7d-fusio...
|Protein||Name: 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|
Recombinant expression: No
|Mass||Experimental: 500 kDa|
|Source||Species: Human immunodeficiency virus 1 / virus / ヒト免疫不全ウイルス 1|
|Source (engineered)||Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / |
+Component #2: protein, HIV-1 Integrase, Sso7d chimera
|Protein||Name: HIV-1 Integrase, Sso7d chimera|
Details: Sso7d protein fused to the integrase N-terminus via an 11-glycine linker
Recombinant expression: No
|Source (engineered)||Expression System: Human immunodeficiency virus 1 / virus / ヒト免疫不全ウイルス 1|
+Component #3: protein, Integrase binding domain of LEDGF/p75
|Protein||Name: Integrase binding domain of LEDGF/p75 / Recombinant expression: No|
|Source (engineered)||Expression System: Homo sapiens / human|
+Component #4: nucleic-acid, DNA (11-MER)
|Nucleic-acid||Name: DNA (11-MER) / Class: DNA / Structure: OTHER / Synthetic: No|
+Component #5: nucleic-acid, DNA (23-MER)
|Nucleic-acid||Name: DNA (23-MER) / Class: DNA / Structure: OTHER / Synthetic: No|
ACTGCTAGAG ATTTTCCACA CTG
+Component #6: nucleic-acid, DNA (37-MER)
|Nucleic-acid||Name: DNA (37-MER) / Class: DNA / Structure: OTHER / Synthetic: No|
CAGTGTGGAA AATCTCTAGC AGTTACAGTC AGCGTAC
|Sample solution||Specimen conc.: 0.5 mg/ml / pH: 8|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277 K / 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.
-Electron microscopy imaging
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 95 e/A2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 22500 X (nominal), 38167 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 90 - 90 K)|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Image acquisition||Number of digital images: 1598 / Sampling size: 5 microns|
Details: Individual frames were gain-corrected, aligned, and summed with the application of an exposure filter using MotionCor2, according to the nominal dose rate.
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 11099|
|3D reconstruction||Algorithm: FOURIER SPACE / Software: Frealign / CTF correction: performed internally in Relion and Frealign / Resolution: 4.6 A / Resolution method: FSC 0.143 CUT-OFF / Details: Resolution-limited refinement used throughout / Euler angles: Frealign 3D classification and refinement|
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