HYBRID NMR/CRYO-EM STRUCTURE OF THE HIV-1 RNA DIMERIZATION SIGNAL

Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach.

RNA prepared by in vitro transcription using T7 RNA polymerase

in vitro transcription vector pT7-TP(deltai)

GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER

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

NMR studies conducted with multiple samples prepared by differential nucleotide-specific 2H labeling.

protonated A-H2 and A,G-ribose carbons; all other sites deuterated

protonated A-H2,H8 and fully protonated G; all other sites deuterated

protonated G-H8, C-H6,ribose; all other sites deuterated

Samples in D2O, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, Tris, pH 7.2, T= 308K

Used GB for solvent simulation, NOEs and EM data simultaneously as restraints

400 uM Fully protonated, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, Tris, 100% D2O

400 uM protonated A-H2 and A,G-ribose carbons A2rGr; all other sites deuterated A2rGr, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, 100% D2O

400 uM protonated A-H2,H8 and fully protonated G; all other sites deuterated A28GH, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, 100% D2O

400 uM protonated G-H8, C-H6,ribose; all other sites deuterated G8C6r, 5 mM NaCl, 140 mM KCl, 1 mM MgCl2, 100% D2O

Case, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, and Kollman

RNA INERNAL LOOPS, SHEARED GA PAIRS, GU WOBBLE, S-TURN THERMODYNAMICS, RNA