REL OBS RCSB RCSB RCSB 2014-07-29 2014-09-24 2015-09-02 2015-09-02 2015-09-02 Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer 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 ESCRT-III copolymer of IST1 and CHMP1B ESCRT-III copolymer of IST1 and CHMP1B 2-stranded helical filament composed of one strand of IST1 subunits and one strand of CHMP1B subunits 2 IST1 (Increased Sodium Tolerance 1) Homo sapiens Human
Uniprot Isoform 4 P53990-4 (IST1 homolog, Homo sapiens), residues 1-189
true Escherichia coli BL21 pGEX-2T-TEV P53990 Charged multivesicular body protein 1b Homo sapiens Human
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 Escherichia coli BL21 pGEX-2T-TEV Q7LBR1 helical helicalArray 8.0
25 mM Tris, pH 8.0, 25 mM NaCl
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).
 ETHANE 75 77 FEI VITROBOT MARK I Blotted 7-9 seconds (-2 mm offset) and plunge-frozen FEI TECNAI F20 FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 200 2.0 GATAN LIQUID NITROGEN 2012-12-08 KODAK SO-163 FILM NIKON SUPER COOLSCAN 9000 10 Gatan 626 side entry FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 2.7 FEI TITAN KRIOS AUTOGRID HOLDER 2013-04-22 FEI FALCON I (4k x 4k) NIKON SUPER COOLSCAN 9000 15 JEOL 3200FSC FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 3.4 JEOL 3200FSC CRYOHOLDER in-column JEOL Omega 2013-05-09 DIRECT ELECTRON DE-12 (4k x 3k) NIKON SUPER COOLSCAN 9000 20
Iterative Helical Real Space Reconstruction (IHRSR) single particle algorithm as implemented in SPIDER. High resolution refinement in RELION.
2.96 21.06 C1 OTHER 3.6 OTHER CTFFIND3, EMAN2, SPIDER, 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.
per particle as implemented in RELION
 emd_6005.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 340 340 340 0 0 0 340 340 340 408.00003 408.00003 408.00003 90.0 90.0 90.0 X Y Z -7.89661789 8.43594933 0.00521642 0.72658151 1.2 1.2 1.2 1.6 AUTHOR Reconstruction of the IST1-CHMP1B ESCRT-III copolymer
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 3FRR A FLEXIBLE FIT Coot, Chimera, NAMD MDFF, Rosetta
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.
Molprobity validation, cross correlation REAL
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