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SUPPLEMENTAL MATERIALS
GTPase activation of elongation factor EF-Tu by the ribosome
during decoding
Jan-Christian Schuette, Frank Murphy, Ann C. Kelley, John Weir, Jan Giesebrecht,
Sean R. Connell, Justus Loerke, Thorsten Mielke, Wei Zhang, Pawel A. Penczek, V.
Ramakrishnan and Christian M.T. Spahn
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Supplementary methods
Preparation of the 70S•tRNA•EF-Tu•GDP•kirromycin complex:
Ribosomes, tRNA and mRNA were prepared as described previously (Selmer et al,
2006). The ribosome initiation complex was formed by addition of mRNA (sequence
5′-GGCAAGGAGGUAAAAAUGUUCAAA-3′ , ordered from Dharmacon) and
deacylated initiator tRNAfMet to 70S ribosomes. The gene for T. thermophilus EF-Tu
was cloned into the T7 expression vector pet 42a, overexpressed and purified first on
a nickel column and then on by gel filtration on Superdex.
Ternary complex (TC) was formed in two steps, the formation of the binary EF-TuGTP complex, followed by the addition of Phe-tRNAPhe. Phenylalanine to 400 μM (all
concentrations listed are final for this TC formation), guanosine triphosphate to 6 mM,
phosphoenolpyruvate to 6mM and adenosine triphosphate to 6mM were mixed. The
pH was then adjusted to 7.5 using 0.1 M KOH. EF-Tu was then added to 165 µM,
and pyruvate kinase (Sigma-Aldrich) to 20 µg/mL. After incubation for 20 minutes at
37˚C, Phe-tRNAPhe and its charging enzymes were added: PheRS to 50 nM, PhetRNAPhe to 30 µM and inorganic pyrophosphatase (Sigma-Aldrich) to 10 µg/mL. The
TC was then incubated a further 20 minutes at 37˚ C.
The initiation complex (IC) was formed in a single non-enzymatic step. T.
thermophilus 70S ribosomes to 1 µM, Z4C mRNA to 3 µM, and tRNAfMet to 1.5 µM
were mixed. The IC was then incubated for 30 minutes at 55˚C, and then cooled on
ice before mixing with the TC.
Immediately before mixing the TC and IC, kirromycin (Sigma-Aldrich) was added to
the IC to a final concentration of 50 µM. The TC was then added to the IC (making
the full complex (FC)) and incubated for 30 minutes at 37˚ C. The FC was then
cooled to room temperature and histidine was added to 1.5 mM. Ribosomal
complexes containing bound his-tagged EF-Tu were purified as described previously
for release factors (Petry et al, 2005).
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Cryo-EM of the 70S•tRNA•EF-Tu•GDP•kirromycin complexes
Ribosomal complexes were diluted to a concentration of 30 nM and subsequently
frozen onto Quantifoil grids using a Vitrobot (FEI) device. Micrographs where
collected on a Tecnai G2 Polara (FEI) at 300 kV and 39,000x magnification under low
dose conditions (19 e-/Å2) and scanned on a D8200 Primscan drum scanner
(Heidelberger Druckmaschinen) with a step size of 4.758 m corresponding to 1.26
Å on the specimen scale.
The Contrast Transfer Function (CTF) defocus values for the micrographs were
determined with CTFind (Mindell & Grigorieff, 2003). Ribosomal projection images
were automatically identified using the program Signature (Chen & Grigorieff, 2006)
and were subsequently screened visually or automatically. From the selected
projections, a reconstruction was generated by projection matching procedures and
refined using the SPIDER software package (Frank et al, 1996). The complete data
set comprised 586,329 projection images collected from 452 micrographs at a 0.6 –
4.0 µm defocus range.
After multi-particle refinement (Connell et al, 2008; Penczek et al, 2006), which was
performed with 3-times or 2-times decimated pictures (see below) the data set of the
major subpopulation I (323,688 projection images) was further refined at full image
size. During the later refinement rounds, positivity of the reference volumes was
enforced, the power spectrum of the cryo-EM map was scaled to the power spectrum
of a model density derived from the atomic coordinates of the X-ray structure of the
70S ribosome (Selmer et al, 2006) and the map was subsequently low-pass filtered
according to the current resolution estimate.
Rigid body docking of atomic models was first done manually using O (Jones &
Kjeldgaard, 1997) and then automatically using the SITUS software package
(Wriggers et al, 1999) or SPIDER. Final figures were prepared with UCSF Chimera
(Pettersen et al, 2004) or Pymol (DeLano Scientific).
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Multi-particle refinement
Initially,
a
classical
single
particle
reconstruction
of
the
T.
thermophilus
70S•EFTu•Phe-tRNA•GDP•kirromycin complex (163,000 particle images, 7.8 A
resolution with the
0.5FSC
criterion) was obtained with a previous cryo-EM
reconstruction of the vacant E. coli 70S ribosome as initial reference. However,
although the specimen behaved quite well during initial phases of image
reconstruction and always exhibited strong density for the ternary complex, the maps
showed indications of conformational heterogeneity as we progressed to subnanometer resolution For example, the stalk base region was essentially disordered
and also the L1 protuberance had a fragmented appearance. In order to account for
conformational
heterogeneity
of
the
T.
thermophilus
70S•EFTu•Phe-
tRNA•GDP•kirromycin complex the data set was gradually increased to 586,329
particle images and sorted into five “subpopulations” using the “multi-particle
refinement” (Penczek et al., 2006; Connell et al., 2008) technique.
In this approach, an increasing number of references is used for projection matching,
thereby assigning fractions of the former uniform data set to new “subpopulations” of
particle images corresponding to different conformations. Multi-particle refinement
does not require prior information about the nature of the structural variability of the
sample as an iterative refinement regime secures that the particles assigned to a
subpopulation are used for a proper reconstruction. It therefore avoids the
disadvantages of supervised classification strategies with their potential bias towards
the references that are applied.
In each sorting step, we increased the number of references by adding the cryo-EM
reconstruction of the vacant E. coli 70S ribosome (Supp. Fig. 1, grey box on top row,
depicted in both side and top view) as a new reference to the already refined
reconstructions. Iterative refinement in one sorting step was continued until stable
angular assignment and subpopulations were obtained (less than 5% of the particle
images changed their angular assignment). In addition to the major subpopulation
two more “meaningful” reconstructions obtained from subpopulations II and III were
obtained, when four reference structures were used. Further sorting with five
reference structures did not increase the number of biologically meaningful
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structures, but resulted in additional reconstructions, which showed signs of
reconstruction artefacts probably caused by the limited amount of particle images
(populations IV and V). Therefore, further multi-particle refinement was not
attempted.
Interestingly, in comparison to the input reference structure from E. coli, the
structures yielded by the multi-particle refinement clearly resemble distinct features of
T. thermophilus ribosomes, e.g. a different conformation of the 30S subunit spur. This
finding strengthens the case for multi-particle refinement as an unsupervised
classification approach that avoids strong bias towards the references used for
sorting.
Two major conformational changes can be observed when the cryo-EM map of major
subpopulation I is compared to the structures computed from subpopulation II and III,
respectively (supplemental Figure S2). In the cryo-EM map of subpopulation II a
counter-clockwise rotation of the small ribosomal subunit versus the large ribosomal
subunit is present (supplemental Figure S2A), which is similar to the ratchet-like
subunit rearrangement (RSR) promoted by EF-G/eEF2 (Connell et al., 2007; Frank
and Agrawal, 2000; Spahn et al., 2004; Valle et al., 2003b).
A conformational change of the stalk-base region is revealed when the cryo-EM map
of subpopulation III is compared to the structure of the major subpopulation I
(supplemental Figure S2B). In the main population the stalk base, i.e. helices H43
and H44 of 23S rRNA has moved inwards – which is in agreement with a previously
published cryo-EM map of the E. coli ribosomal decoding complex (Valle et al., 2002;
Valle et al., 2003a). However, in the reconstruction calculated from subpopulation III
of the data set, the stalk-base region adopts an outer position, similar to “vacant”
ribosomes (supplemental Figure S2B, red circle). Therefore, the inward movement of
the stalk base region is not necessarily coupled to ternary complex binding as
previously assumed.
A detailed analysis of the conformational modes requires further work and will be
presented elsewhere.
5
Supplemental Results and Discussions
Molecular interpretation of the cryo-EM map
The crystal structure of the 70S ribosome from T. thermophilus in complex with
mRNA and tRNAs was recently solved at 2.8 Å resolution (Selmer et al., 2006),
which allowed a direct comparison to our cryo-EM map. A satisfying fit could be
obtained by docking the atomic model of the 70S ribosome (Selmer et al., 2006) as
three rigid bodies corresponding to the main body of the 50S subunit and the head
and the body/platform domains of the 30S subunit. These major domains of the
ribosome are known to be movable relative to each other (Connell et al., 2007; Frank
and Agrawal, 2000; Schuwirth et al., 2005; Spahn et al., 2001; Valle et al., 2003b),
and only small translational movements (1-2 Å) were necessary to fit the atomic
structures of the domains into the density. The only elements that required further
adjustment were the L1 stalk and the L7/L12 stalk base region of the 50S subunit,
which are known to be dynamic (Connell et al., 2007; Harms et al., 2001; Schuwirth
et al., 2005; Selmer et al., 2006; Valle et al., 2003a; Yusupov et al., 2001).
Interestingly, density for ribosomal protein L36 is present in our cryo-EM density map
(supplemental Figure S4). L36 was not observed in the 2.8 Å crystal structure of the
70S ribosome from T. thermophilus (Selmer et al., 2006); however, it was modeled in
another X-ray structure of the T. thermophilus 70S ribosome at 3.7 Å resolution
(Korostelev et al., 2006), although cross-crystal averaging of the two 70S data sets
indicated that L36 should be absent also in the latter map (Simonovic and Steitz,
2008). It is not clear why L36 is missing in the X-ray structures of the 70S ribosomes
from T. thermophilus. However, our cryo-EM map clearly shows that 70S ribosomes
from T. thermophilus in solution do contain protein L36.
In order to quantify the fit, we calculated a local cross-correlation map between the
cryo-EM map and density derived from the atomic coordinates (supplemental
Fig. S5). For most parts of the map a local cross-correlation coefficient of 0.7 or
higher indicates a good agreement between the atomic model and the cryo-EM map
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consistent with a good visual fit. Regions of poor correlation include the L1 stalk and
the L7/L12 stalk base region. Although the appearance of density for these highly
dynamic parts of the ribosome improved due to our multi-particle refinement, both
these regions contain fragmented density (not shown) indicating local conformational
heterogeneity. Some regions of poor correlation are due to the incompleteness of the
atomic model. For example, density is present in the cryo-EM map that probably
corresponds to the Shine-Dalgarno sequence at the 5 end of mRNA interacting with
the 3 end of 16S RNA (supplemental Fig. S5), which was disordered in a 2.8 Å
crystal structure (Selmer et al., 2006). The analysis of the local correlation map is
therefore a useful tool to detect small local differences. However, in general, the
conformational changes appear to be small and we did not attempt to locally distort
the molecular model within domains because we felt such local distortions of crystal
structures require even higher resolution in the absence of corroborating evidence.
Comparison of the cryo-EM density with X-ray structures of EF-Tu
The crystal structures of T. thermophilus EF-Tu with the GTP analog GMPPNP
(Berchtold et al., 1993) or methylkirromycin (Vogeley et al., 2001) have been solved.
The two structures are not equivalent: in the methylkirromycin structure, domains 2
and 3 are rotated relative to domain 1 by about 10° and 15°, respectively (Vogeley et
al., 2001). When we compared the two structures to our cryo-EM density, it became
clear that the EF-Tu•GDP•methylkirromycin structure (Vogeley et al., 2001) could be
docked readily into the cryo-EM density (Fig. 2), whereas the EF-Tu•GMPPNP
structure (Berchtold et al., 1993) could not be fit into the density as a single rigid
body. When the three domains of EF-Tu in this structure were fit individually into the
density, the resulting modified structure agreed well with the EFTu•GDP•methylkirromycin structure, with a r.m.s.d. value of 0.76 Å for the backbone
atoms (excluding the two switch regions). In light of our resolution, this difference is
well within the error of the data since the r.m.s.d. value for the individual domains of
the two crystal structures is already 0.4 to 0.5 Å (Vogeley et al., 2001). This
comparison demonstrates the accuracy of docking individual domains at this
resolution.
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Supplemental Figure Legends
Supplemental Figure 1: Multi-particle refinement strategy
In order to account for conformational heterogeneity of the T. thermophilus 70SEFTuPhe-tRNAGDPkirromycin complex the multi-particle refinement was used to
sort the data set into up to five subpopulations (Penczek et al., 2006; Connell et al.,
2008). During the refinement the number of reference structures was successively
increased by adding the structure of a vacant E. coli 70S ribosome as a new
reference. The respective cryo-EM maps of the ribosomal complexes are depicted in
the side view. Numbers and percentages indicate total particle projections and
fractions of the whole data set, respectively. Population I embodies the majority of
particle images and represents the cryo-EM map analyzed in this study.
Supplemental Figure 2: Comparison of populations II and III with the main
conformation
(A) Superposition of Population I (gold) compared to Population II (pink, transparent).
The reconstructions (aligned on the 50S subunit) are shown from the top with the
50S subunit above the 30S subunit. An arrow indicates a ratchet-like subunit rotation
of the 30S subunit versus the large subunit. (B) Superposition of Population I (gold)
compared to Population III (blue, transparent). The reconstructions are shown from
the side. The stalk base of population III is abbreviated (red circle).
Supplemental Figure 3: Resolution curve.
The Fourier Shell Correlation (FSC) vs. spatial frequency plot is shown. The
resolution of the cryo-EM map is 6.4, 5.7, and 4.7 Å as estimated from the 0.5 FSC,
0.143 FSC (Rosenthal and Henderson, 2003) and 3 sigma criteria (Orlova et al.,
1997), respectively. The resolution was estimated using a generous mask with soft
edges in order to remove external noise.
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Supplemental Figure 4: Resolution of Secondary Structure elements and
extended protein chains
Close-ups of various regions of the cryo-EM map of the T. thermophilus 70SEFTuPhe-tRNAGDPkirromycin complex at 6.4 Å resolution (grey mesh) superposed
with fitted of molecular models (coloured ribbons):
(A) Protein S7.
(B) Protein S12.
(C) Protein L36.
Supplemental Figure 5: Comparison of the cryo-EM reconstruction with the
docked molecular models by local cross-correlation coefficient.
Surface rendering of the cryo-EM reconstruction of the 70SEF-TuPhetRNAGDPkirromycin complex is shown color-coded by the local cross-correlation
coefficient (CCC) between the cryo-EM density and our molecular model (converted
to density and low pass filtered to the resolution of the cryo-EM map). The local CCC
was calculated within a moving box (size 9x9x9 pixels at 1.26 Å pixel size) in steps of
4 pixels.
(A, B) The ribosomal 30S subunit is shown from the 50S side (A) and the solvent side
(B).
(C, D) The ribosomal 50S subunit is shown from the 30S side (C) and the solvent
side (D).
(E, F) The ribosomal ligands are shown from two different orientations.
Colour code for CCC: light blue: > 0.7, white/blue: > 0.6, yellow: > 0.5, orange: > 0.4,
red: < 0.4. Prominent ribosomal landmarks are indicated as in Figure 1. Moreover
some regions with poor local CCC are labeled (for more details see supplemental
text). The figure was prepared with UCSF chimera (Goddard and Ferrin, 2007).
Supplemental Movie 1: Overview of the 70SEF-TuPhe-tRNAGDPkirromycin
complex.
A surface representation of the cryo-EM map is shown rotating around an axis
perpendicular to the intersubunit plane. Colour code: yellow: 30S subunit, blue: 50S
subunit, red: EF-Tu.GTP, pink: A/T-side Phe-tRNA, green: peptidyl-site tRNA,
orange: exit-site tRNA.
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Supplemental Movie 2: Interaction of the SRL with the GTPase center of EF-Tu.
A simulation depicting a putative conformational change of the switch 2 region of EFTu between the GTP conformation (Berchtold et al., 1993) and the putative GTPase
conformation (Vogeley et al., 2001). Molecular models fitted: EF-Tu.GDP.aurodox
(red cartoon), EF-Tu.GMPPNP (green cartoon). His85 is labelled (red arrow). For
further details see legend of Fig. 6A in the main text.
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