Journal: Science / Year: 2017 Title: A supramolecular assembly mediates lentiviral DNA integration. Authors: Allison Ballandras-Colas / Daniel P Maskell / Erik Serrao / Julia Locke / Paolo Swuec / Stefán R Jónsson / Abhay Kotecha / Nicola J Cook / Valerie E Pye / Ian A Taylor / Valgerdur ...Authors: Allison Ballandras-Colas / Daniel P Maskell / Erik Serrao / Julia Locke / Paolo Swuec / Stefán R Jónsson / Abhay Kotecha / Nicola J Cook / Valerie E Pye / Ian A Taylor / Valgerdur Andrésdóttir / Alan N Engelman / Alessandro Costa / Peter Cherepanov / Abstract: Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the ...Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.
Details: 1 M NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl, pH 6.5
Grid
Model: PELCO Ultrathin Carbon with Lacey Carbon / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV Details: To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4-ul drop of 200 mM NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 and ...Details: To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4-ul drop of 200 mM NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 and blotted again for 2.5 s followed by plunging into liquid ethane..
Details
A 4 ul drop of freshly prepared intasome in 1 M NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 was applied onto glow-discharged lacey carbon grids coated with ultrathin carbon (product 01824, Ted Pella). The grids were incubated for 30 s under 100% humidity in a Vitrobot Mark IV (FEI) at 20 oC. To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4 ul drop of 200 mM NaCl, 3 mM CaCl2, and 25 mM BisTris-HCl pH 6.5 and blotted again for 2.5 s, followed by plunging into liquid ethane.
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Electron microscopy
Microscope
FEI POLARA 300
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
5M0Q model was docked to new map (updated relion version and pixel size corrected) in Chimera and refined using phenix.real_space refine and interactively adjusted in coot.
Refinement
Space: REAL / Protocol: OTHER / Overall B value: 100 / Target criteria: correlation coefficient
Output model
PDB-7zpp: Cryo-EM structure of the MVV CSC intasome at 4.5A resolution
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