2014-07-09 OBS 2013-06-25 PDBe PDBe 2013-07-24 2014-07-09

Past the one year on hold period.

Cheung M, Shen D, Makino F, Kato T, Fernando VK, Roehrich D, Martinez-Argudo I, Walker ML, Liu X, Brown J, Frazer G, Mantell J, Mina P, Todd T, Sessions RB, Namba K, Blocker AJ Tip complex, Type III secretion system, Shigella flexneri, Q51A Cheung M, Shen D, Makino F, Kato T, Fernando VK, Roehrich D, Martinez-Argudo I, Walker ML, Liu X, Brown J, Frazer G, Mantell J, Mina P, Todd T, Sessions RB, Namba K, Blocker AJ Structure and activation mechanism of the type III secretion system needle tip complex To Be Published Veenendaal AK, Hodgkinson JL, Schwarzer L, Stabat D, Zenk SF, Blocker AJ The type III secretion system needle tip complex mediates host cell sensing and translocon insertion MOL.MICROBIOL. 63 1719 1730 2007 17367391 doi:10.1111/j.1365-2958.2007.05620.x Blocker AJ, Deane JE, Veenendaal AK, Roversi P, Hodgkinson JL, Johnson S, Lea SM What's the point of the type III secretion system needle? PROC.NAT.ACAD.SCI.USA 105 6507 6513 2008 18458349 doi:10.1073/pnas.0708344105 emd_2406_fsc.xml 2 Conformation of the tip complex from the type III secretion system of Shigella flexneri bound to the needle with MxiH mutation Q51A Five subunits of the tip complex bound to fifty subunits of the needle 0.68

Tip complexes were analysed from purified needle complexes and therefore when still bound to the needle tip

cellular-component Tip complex 0.21 true Shigella flexneri Heteropentameric 1 serotype 5a IPR006135 GO:0030257 Extracellular component of membrane embedded complex Type III secretion system pACT3, pBAD M90T derivative Shigella flexneri cellular-component Needle 0.47 Shigella flexneri 1 true serotype 5a IPR006135 GO:0030257 Type III secretion system Extracellular component of membrane embedded complex pACT3, pBAD Shigella flexneri M90T derivative NONE NONE Thon rings visualised at 50-100,000 times magnification using the fast FT and corrected accordingly LAB6 9 BRIGHT FIELD 460 1400 1400 FLOOD BEAM FEI single tilt holder FEI EAGLE (4k x 4k) 2 70754 FEI TECNAI 20 31-AUG-2011 0 SIDE ENTRY, EUCENTRIC 200 50000 3.56 250 15 16

Residues comprising the N domain were removed. The 10 residues at the C terminus (not resolved in the crystal structure) were built into an alpha helix

Chimera rigid body REAL 2J0N A Carbon coating mica sheets were submerged in a drop of 0.01% (w/v) DDM until the carbon sheet partially detached from the mica. The mica was then submerged in the sample for 5 s, then submerged in a drop of water for 5 s. The mica was then placed onto the surface of a drop of 1% uranyl acetate and allowed to sink, causing the carbon to float on the surface of the drop. After 10 s, the carbon sheet was adsorbed onto a grid. particle 0.1 300 mesh copper grid with a thin carbon film to which the specimen was adsorbed to

0.1% (w/v) DDM, 150 mM NaCl, 25 mM Tris pH 8, 5 mM EDTA

8 singleParticle Projection matching SPIDER: theta 4 degrees EMAN2, SPIDER, bespoke-2D-alignment, bespoke-X-and-Y-shifting First zero set to 18 angstrom, no CTF correction not required 22.5 Data set divided into two sets and reconstruction parameters applied to both sets. FSC determined and resolution determined at 0.5 cut-off. 127 C1 2545

Particles were selected using helix boxer from EMAN2. After alignment, Euler angles were determined by projection matching with projections of a previously determined 16A resolution map of the needle. The reconstruction was iterated and helical pattern searching and imposition was applied to each new reference generated after each iteration.