Structure of tetrameric HIV-1 Strand Transfer Complex Intasome

Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.

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.

Resolution-limited refinement used throughout

performed internally in Relion and Frealign

complex formed by a tetrameric assembly of Sso7d-fusion HIV-1 Integrase with the product of DNA strand transfer

Individual frames were gain-corrected, aligned, and summed with the application of an exposure filter using MotionCor2, according to the nominal dose rate.

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.

MGSSHHHHHHSSGLVPRGSHMATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLE
KQKKGGGGGGGGGGGFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDC
THLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNP
QSQGVIQSMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRV
YYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED

(DA)(DC)(DT)(DG)(DC)(DT)(DA)(DG)(DA)(DG)(DA)(DT)(DT)(DT)(DT)(DC)(DC)(DA)(DC)(DA)
(DC)(DT)(DG)

(DC)(DA)(DG)(DT)(DG)(DT)(DG)(DG)(DA)(DA)(DA)(DA)(DT)(DC)(DT)(DC)(DT)(DA)(DG)(DC)
(DA)(DG)(DT)(DT)(DA)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DC)(DG)(DT)(DA)(DC)

National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)

Leona M. and Harry B. Helmsley Charitble Trust Grant

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

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

integrase, integration, transposase, transesterification, VIRAL PROTEIN

FLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVA
SGYIEAEVIPAETGQETAYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNKELKK
IIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRDPVWKGPAK
LLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED