Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit

Determination of the ribosome structure to a resolution of 2.5 Å by single‐particle cryo‐EM

“…Interestingly, some residue densities reveal inconsistencies with the residues from the T. cruzi sequence in the database, a reflection of natural mutations. For example, in our structure, residue 64 in protein eL37 is a Cys in the sequence of the database, but clearly a Met based on the density . The resultant atomic model of the large ribosomal subunit was subjected to real‐space refinement using PHENIX against the map generated by sharpening with the negative B factor of −50 Å 2 .…”

“…With traditional recording devices, the best resolution for asymmetric molecules such as ribosomes was not better than 5 Å . It has now moved into the range of 2–3 Å, permitting de novo atomic modeling . Improvements in the data quality of cryo‐EM have been provided primarily by advances in instrumentation.…”

“…The ribosome has served as a benchmark sample for decades in the development of single‐particle cryo‐EM . Sub‐3 Å resolution of ribosomes, in the range of 2.5–2.9 Å, has now been achieved by four groups . Our own 2.5‐Å cryo‐EM reconstruction is of the ribosome extracted from Trypanosome cruzi , a protozoan pathogen causing Chagas disease in humans.…”

“…1,2 It has now moved into the range of 2-3 Å , permitting de novo atomic modeling. [3][4][5][6] Improvements in the data quality of cryo-EM have been provided primarily by advances in instrumentation. First of all, the direct electron detector device (DDD) dramatically improves the Detection Quantum Efficiency (DQE) and allows fractionating the electron dose of each micrograph.…”

Advances in the field of single-particle cryo-electron microscopy over the last decade

“…(For the record it should be mentioned, though, that the “near-atomic resolution” in the title referred to a value around 4.5Å, better by just 1Å than the film-based study of Hashem et al 9 which appeared at the same time, obtained from 160,000 particles, rather than millions). Since then numerous ribosome structures from both prokaryotes and eukaryotes have appeared in the 3Å range, the most recent one being the structure of the large subunit from T. cruzi solved in my lab 14 at 2.5Å, a resolution sufficient to show water molecules as well as rRNA modifications, and to allow de novo atomic modeling. Most notable in this recent “resolution revolution” has also been the achievement of close-to-atomic resolution for small membrane-bound proteins, starting with the elucidation of the TRVP1 channel by the group of Yifan Chen at the University of California at San Francisco 15,16 .…”