2015-09-02 OBS 2014-07-29 RCSB RCSB 2014-09-24 2015-09-02 2015-09-02

Author will re-deposit the map.

McCullough J, Colf LA, Saunders MG, Arthur C, Sundquist WI, Frost A ESCRT-III, IST1, CHMP1B, cytokinesis, membrane fission McCullough J, Colf LA, Saunders MG, Arthur C, Sundquist WI, Frost A Structures of Helical and Spiraling ESCRT-III Filaments Suggest a Model for Membrane Constriction To Be Published

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2 ESCRT-III copolymer of IST1 and CHMP1B 2-stranded helical filament composed of one strand of IST1 subunits and one strand of CHMP1B subunits protein IST1 (Increased Sodium Tolerance 1) hIST1, Putative MAPK-activating protein PM28

Uniprot Isoform 4 P53990-4 (IST1 homolog, Homo sapiens), residues 1-189

Homo sapiens Human true P53990 Escherichia coli BL21 pGEX-2T-TEV protein Charged multivesicular body protein 1b CHMP1.5 Chromatin-modifying protein 1b Short name=CHMP1b Vacuolar protein sorting-associated protein 46-2 Short name=Vps46-2 Short name=hVps46-2

CHMP1B cDNA was originally obtained from HeLa cDNA. Originally reported in von Schwedler, U. K. et al. The protein network of HIV budding. Cell 114, 701-713 (2003). It contains a K34E mutation compared to GenBank Accession number AAG01449.

true Homo sapiens Human Q7LBR1 pGEX-2T-TEV Escherichia coli BL21 Blotted 7-9 seconds (-2 mm offset) and plunge-frozen ETHANE 75 FEI VITROBOT MARK I 77 BRIGHT FIELD GATAN LIQUID NITROGEN 2.0 FLOOD BEAM 08-DEC-2012 10 FIELD EMISSION GUN FEI TECNAI F20 KODAK SO-163 FILM 200 Gatan 626 side entry 15 FEI FALCON I (4k x 4k) FLOOD BEAM FEI TITAN KRIOS 300 BRIGHT FIELD FIELD EMISSION GUN 2.7 FEI TITAN KRIOS AUTOGRID HOLDER 22-APR-2013 JEOL 3200FSC FIELD EMISSION GUN BRIGHT FIELD FLOOD BEAM DIRECT ELECTRON DE-12 (4k x 3k) 300 3.4 09-MAY-2013 20 in-column JEOL Omega JEOL 3200FSC CRYOHOLDER NIKON SUPER COOLSCAN 9000 Molprobity validation, cross correlation

3FRR was docked manually into the segmented density using Chimera. Regions with poor fit to density were identified using the Rosetta loops-from-density algorithm and iteratively fitted using alternating cycles of Rosetta's rebuild and refine protocol and manual refinement in Coot. The full ring of IST1 structures was then refined using Rosetta's symmetry constraints. Finally, backbone hydrogen bonds in the helical regions were constrained and two cycles of loop rebuilding with constraints were performed. The IST1 model was then combined with the CHMP1B model to form a heterodimer and this structure was refined by iterating between manual rebuilding and refinement using MDFF.

Coot, Chimera, NAMD MDFF, Rosetta flexible REAL 3FRR A 2.96 C1 RIGHT HANDED 21.06 helicalArray 3.5 uL of pelleted and resuspended liposome-nucleated IST1NTD-CHMP1B copolymers were applied to glow-discharged Quantifoil holey carbon grids (2 micron hole size, 2-4 micron spacing, 200 mesh). 8.0

25 mM Tris, pH 8.0, 25 mM NaCl

helical IHRSR initially, followed by high resolution asymmetric refinement in RELION, followed by helical averaging in real space CTFFIND3, EMAN2, SPIDER, RELION per particle as implemented in RELION

51 copies (3 complete turns) of the asymmetric RELION reconstruction were transformed according to the helical symmetry, resampled on the original grid, and summed together.

3.6 FSC 0.143, gold-standard

Iterative Helical Real Space Reconstruction (IHRSR) single particle algorithm as implemented in SPIDER. High resolution refinement in RELION.