Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallograhy.
BACKGROUND
Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscopy (cryo-EM) has yielded fairly detailed three-dimensional reconstructions of ribosomes. These were used to assist in the determination of higher resolution structures by X-ray crystallography.


RESULTS
Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at approximately 10 A. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 A, cryo-EM reconstructions were indispensable for analyzing their 7.2 A multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown.


CONCLUSIONS
Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.
Harms
J
J
Max-Planck Research Unit for Ribosomal Structure, Hamburg, Germany.
Tocilj
A
A
Levin
I
I
Agmon
I
I
Stark
H
H
Kölln
I
I
van Heel
M
M
Cuff
M
M
Schlünzen
F
F
Bashan
A
A
Franceschi
F
F
Yonath
A
A
D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry*
D012269:Ribosomal Proteins