X-Ray Analysis of Prokaryotic and Eukaryotic Ribosomes - Biophysical Approaches to Translational Control of Gene Expression - page 2

1.1.2 Challenges for X-Ray Studies of Ribosomal Complexes

Crystallographic methods can shed light on many structure-related issues, from
overall molecular conformations and ternary and quaternary interactions to second-
ary structure information and details about atomic bonds. In contrast to NMR and
Cryo-EM approaches, there is no limitation to the size of molecule or assembly to
be studied.
The main bottleneck in crystallographic studies is that a well-diffracting crystal
must be found, and thus the information gleaned about the dynamic nature of the
molecules to be studied will be very limited from only a single diffraction experi-
ment. In other words, the price to pay for the high accuracy of X-ray crystallo-
graphic structures is that the method is very time-consuming.
For the ribosome as a huge complex consisting more than 50 components, it is
very important to ensure that the samples are homogenous for crystallization to suc-
ceed. In our studies we have achieved this by two different strategies: For the
prokaryotic studies we have chosen to work with a thermophilic bacteria because
the ribosomes isolated from this organism are more robust and resistant to degrada-
tion. For the eukaryotic ribosome very gentle isolation protocols were developed to
ensure that all the ribosomal components are intact and present. We exploited the
observations that glucose starvation of the growing yeast cells leads to inhibition of
initiation and accumulation of very homogenous ribosomes without any ligands
(Ashe et al. 2000 ) .
A further complication arises since ribosome crystals, as typically seen in RNA
crystallography, diffract only poorly which results in electron density maps that are
imprecise and difficult to interpret. Therefore special care has to be taken during
post-crystallization treatment to avoid damaging the crystals (i.e., when transferring
cryo-protection) and even for the freezing process itself we only use the most robust
methods of freezing directly in the gaseous N 2 stream at 100 K rather than plunging
into liquid N 2 , ethane, or propane as is common practice in X-ray structural projects.
A combination of severe radiation decay and generally weak diffracting power lim-
its the amount of data that can be collected from each crystal making it necessary to
merge data collected on different crystals to obtain complete datasets which invari-
antly degrades the data quality.

1.2 Crystal Structures of Prokaryotic Ribosome Complexes

1.2.1 Introduction

Translation of nucleotide sequence information in the form of mRNA codons into
amino acids lies at the heart of protein biosynthesis. This process is accomplished by
tRNA molecules that act as adaptors between the mRNA codon and the amino acids
they code for. For accurate protein synthesis, the ribosome is required to position the