Structure of the SecY protein translocation channel in action

Structure of the SecY channel during initiation of protein translocation.

SOME RESIDUES IN THIS ENTRY ARE NOT PROPERLY LINKED.

SEVERAL AMINO ACID RESIDUES IN THIS ENTRY HAVE INCORRECT STEREOCHEMISTRY AT THEIR CA CHIRAL CENTERS.

REFINEMENT PROTOCOL--FLEXIBLE DETAILS--A- and P-site tRNAs from T. thermophilus ribosome structure. mRNA from this structure also used for some modeling steps.

The structure was solved twice: first with a model starting from a 25-Angstrom filtered E. coli ribosome map generated in house, and then a second time using a filtered ribosome model (EMD-5036). In each case, after convergence, maps from two EMAN2 refinements with different parameters were averaged after alignment in Chimera. Four maps in total were averaged to reduce the noise. Resolution method was FSC at 0.5 cut-off for a comparison between the full experimental 3D density map and a calculated map of the docked E. coli ribosome model (this map was calculated to 7 Angstrom resolution with EMAN).

50 mM Tris-acetate, 10 mM Mg(OAc)2, 80 mM KOAc, 0.06% DDM

active ribosome-nascent chain-SecYEG complex

Low dose imaging: automated single particle data collection program from TVIPS was used.

400 mesh Quantifoil holey grids with 2/1 or 1.2/1.2

Blot 1-2 seconds before plunging into liquid ethane (FEI VITROBOT MARK III).

map of active ribosome with a nascent chain inserted into the open SecYEG channel

Docked into the 30S small ribosomal subunit

Docked into the 50S large ribosomal subunit

chains B and C are the A- and P-site t-RNAs docked into the map

EM map of closed SecYEG channel bound to the non-translocating 70S ribosome.

SECY, SECE, SECG, NC100, 50S ribosomal protein L1, 50S ribosomal protein L23P, 50S ribosomal protein L24P, 50S ribosomal protein L29P
70S, preprotein translocase, SECYEG, protein translocation channel, nascent chain, RIBOSOME-PROTEIN TRANSPORT complex