Supplementary Methods
X-ray and SAXS
An initial polyalanine model of the two molecules in the asymmetric unit was build
into the 3.5 Å resolution density map of eEF3-ADP phased by an KAu(CN)2 derivative1. This
model was used for molecular replacement with the 2.4 Å selenomethionine dataset collected
at the ESRF ID29 beamline with =0.9792 Å as previously described1. Experimental phases
derived from the weak anomalous signal were then combined with the model phases, and a
new polyalanine model constructed by RESOLVE2. The model was rebuild in “O”3 and
refined with CNS4 in an iterative manner (Suppl. Table 1). Crystals of apo-eEF3 and
ADPNP-eEF3 were obtained using the same approach as eEF3-ADP1 by co-crystallisation in
the presence of 45% ammoniumsulfate at pH 5.2. Data were collected at the ESRF ID29
beamline with =0.9756 Å or at EMBL-DESY BW7A with =0.995 Å for the apo-eEF3 and
ADPNP-EF3, respectively. All these data collected at 100 K were processed with XDS5
(Suppl. Table 1). The eEF3-ADP structure was used for rigid body refinement against these
data and the resulting model was manually refitted in “O” and refined in CNS (Table 1). The
Ramanchandran
statistics
(%
Most
favored/Additionally
allowed/Generously
allowed/Disallowed) for the refined models are: eEF3-ADP 91.1/8.8/0.1/0.1, apo-eEF3
88.2/11.7/0.1/0.1, eEF3-ADPNP 89.9/10.3/0.2/0.0. For details on the Small-angle x-ray
scattering (SAXS) measurements see Suppl. Notes 1.
Sample preparation for cryo-EM
80S ribosomes6 and RNCs were purified as described before, using truncated mRNA
coding for the first 120 amino acids of DPAP-B with an affinity tag in its N-terminus6-8.
eEF3-Xa-His10 was overexpressed in the yeast expression strain WCG and purified using
TALON metal affinity resin (Clontech). 80S ribosome-eEF3 complexes were reconstituted by
incubating approximately 10 pmol of eEF3 and 1 pmol of ribosomes for 15 min at room
temperature and then 10 min on ice in 20 mM HEPES/KOH (pH 7.5), 10 mM Mg(OAc)2, 150
mM KOAc, 1 mM dithiothreitol (DTT), 0.05% Nikkol, 125 mM sucrose, and, when present,
500 µM ADP, ATP or AMP-PNP and 100 µM neomycin in a final volume of 25 µl. For
RNC-eEF3 complexes 10 µgxml-1 cycloheximide was present.
Binding assays were performed using purified RNCs or empty 80S ribosomes with an
excess of purified eEF3 in the presence of ADPNP and neomycin when indicated (RNCn).
After sucrose density centrifugation supernatant (S) and pellet (P) fractions were analyzed by
SDS-PAGE and Coomassie blue stain. Quantitation of eEF3 binding was done by
densitometric determination of band intensity and setting the eEF3 binding in the presence of
ADPNP and neomycin to 100%, while normalizing according to the intensity of ribosomal
protein. For technical reasons, in order to so saturate the emerged signal sequence of the
nascent chains which otherwise leads to biased orientation of the particles, purified trimeric
Sec61 complex in 0.3% desoxyBigCHAPS (Calbiochem) was added8 for the structural study.
EM and processing
Reconstituted RNC-eEF3 complexes were applied on carbon-coated holey grids9
(Quantifoil) in the presence of Sec618, visualized on a 300 kV Polara cryo-microscope (FEI)
and processed using the SPIDER software package as described7. The dataset (98,267
particles) was first sorted10 according to empty6 (32,782) or programmed8 (65,484)
conformational state of the ribosome, followed by sorting according to presence of eEF3.
37,700 particles were used for the final reconstruction at a resolution of 9.9 Å (6.2 Å) based
on Fourier shell correlation (FSC) of masked volumes at 0.5 (3). The resulting map is shown
without the Sec61 density in Fig. 2 and 3 for better clarity (see Suppl. Fig. 6a for complete
map).
Docking into EM density
Docking of X-ray structures and molecular models of eEF3 (based on MJ079611 and
RLI12) and ribosomal proteins and RNA (PDB 1K5X, 1K5Y and 1K5Z)13 was done with IRIS
Explorer, SPIDER14, O15 and ERNA-3D16. The models of the ribosomal proteins rpL11, rpL5,
rpS5, rpS18 (residues 15-83) and rpS20 were adjusted as rigid bodies to fit into the density.
The proteins rpL5 (residues150-232) and rpS18 (residues15-83) are truncated for better
clarity. A model for the tip of the rRNA helix 39 (nucleotides 1354 to 1370) which is
extended with respect to T. thermophilus rRNA was built in ERNA 3D16 by generating an Ahelix of 6 base pairs (G1354-C1360, G1365- U1370) and a tetraloop (A1361-1364).
The molecular model of eEF3 bound to the ribosome was built on the basis of the ATP
model of eEF3 based on MJ079611. After docking the ABC tandem into the density, the
chromodomain was adjusted by a translation of approx. 5 Å and a rotation of approx. 53°
relative to its position in the crystal structure with G762 and G864 serving as hinges
(connecting it to the α-helical subdomain of ABC2). The HEAT domain was subdivided into
two blocks consisting of the first 5 HEAT repeats (residues 1-200) and the following 3 HEAT
repeats (residues 201-333). These blocks and the 4HB domain were docked as individual rigid
bodies into the EM density. In detail, the first 5 repeats of the HEAT domain was flipped by
160° to fit into the density on the head of the 40S subunit. Afterwards, the last three HEAT
repeats were moved for 15Å translation resulting in a contact with rpS18. Finally, the 4HB
was moved approximately 10 Å towards the ABC1 domain. Using the program Situs 17 for
docking of MalK based18 ADP and ATP models for eEF3 (cc of 0.7 for ATP and 0.62 for
ADP state) confirmed our results with models based on MJ079611 and RLI12. Figures were
produced with PYMOL19 and USFC Chimera20.
Suppl. Legends
Suppl. Table 1 Statistics of the crystallographic analysis of eEF3
No cutoffs based on the standard deviation were applied to intensities during merging or
amplitudes during refinement.
1
Previously published in Andersen, C. F. et al. Acta Crystallogr D Biol Crystallogr 60, 1304-
7 (2004).
2
Values in parentheses are for outer shells.
3
Rmerge = (hj=1,N |Ih-Ih(j)| / N x Ih) for the intensity of reflection h measured N times
4
R-factor = h|Fo|-|Fc| / h|Fo|, where Fc is the calculated structure factor scaled to Fo
5
R-free is identical to R-factor on a subset of test reflections not used in refinement
Suppl. Table 2 Molecular contact regions between eEF3 and the 80S ribosome
Abbreviations: p indicates protein; R indicates ribosomal RNA. Amino acid residues and
bases are given in the one letter code with residue #. S.c. and H.m. indicate that the nucleotide
numbers refer to models based on the sequence of Saccharomyces cerevisiae 18S rRNA and
Haloarcula marismortui 5S rRNA, respectively.
Suppl. Figure 1. Sequence of Saccharomyces cerevisiae eEF3. The secondary structure
representation above the sequence is colored according to domains: HEAT domain in blue,
4HB in yellow, ABC1 in red, ABC2 in green and the chromodomain in purple. The residue
letters are colored according to conservation in an alignment of 16 known eEF3 sequences
(Saccharomyces cerevisiae (eEF3A & eEF3B), Candida glabrata, Eremothecium gossypii,
Kluyveromyces lactis, Clavispora lusitaniae, Debaryomyces hansenii, Candida albicans,
Yarrowia lipolytica, Schizosaccharomyces pombe, Pneumocystis carinii, Aspergillus
fumigatus, Aspergillus oryzae, Gibberella zeae, Chaetomium globosum, Neurospora crassa).
Identical residues are designated by dark grey background and conserved (> 85% identical) by
light grey background. The conserved sequence motifs of the ABC superfamily are indicated
with boxes. Note that the Q-loop box in ABC1 does not contain a glutamine, but His496 may
substitute for it.
Suppl. Figure 2. a,
Stereo view of ADPNP bound to eEF3 at the unusual nucleotide
binding site. The electron density of ADPNP is generated from a 2Fo-Fc omit map contoured
at 1.5 . Residues interacting with ADPNP are shown as sticks. b, The unusual nucleotide
binding site of eEF3 compared to the canonical site modeled form the structure of RLI. The
canonical ADP is prevented from binding to this site by the overlap with Phe35 (shown as
sticks). c, The sulphate binding site formed by the signature motif (shown in green). The
electron density of the sulphate ion is generated from a 2Fo-Fc omit map contoured at 1.5 .
Suppl. Figure 3. Multiple alignment of eEF3 sequences from Saccharomyces cerevisiae
(eEF3A and eEF3B), Candida glabrata, Eremothecium gossypii, Kluyveromyces lactis,
Clavispora lusitaniae, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica,
Schizosaccharomyces pombe, Pneumocystis carinii, Aspergillus fumigatus, Aspergillus
oryzae, Gibberella zeae, Chaetomium globosum, Neurospora crassa.
Suppl. Figure 4. SAXS analysis of eEF3. a-b, Background-subtracted SAXS data of eEF3 at
pH 7.2 (a) and pH 5.2 (b) as a function of concentration before rebinning. From top: 10
mg/ml (red), 5 mg/ml (blue) and 2.5 mg/ml (green). c, Comparison of the two 10 mg/ml data
sets of eEF3 at pH 7.2 (red) and 5.2 (blue) after logarithmic rebinning. d, Distance
distribution functions obtained by GNOM for the two 10 mg/ml data sets of eEF3 at pH 7.2
(red) and 5.2 (blue). Dmax is respectively 125 Å and 110 Å. e-f, Theoretical SAXS curves of
the crystal structure (red), cryo-EM structure (green), and ATP-model (blue) are fitted to the
ph 7.2 (e) and pH 5.2 (f) data for q > qmin = 0.05 Å. g, Average envelope at pH 7.2 with the
ATP-model superimposed. h, Average envelope at pH 5.2 with the crystal structure
superimposed.
Suppl. Figure 5. Nucleotide dependency of eEF3 binding.
a, Binding assay using purified RNCs with an excess of purified eEF3 in the presence of
ADP, ATP or ADPNP, respectively. Presence of neomycin (neo) is indicated. After sucrose
density centrifugation supernatant (s) and pellet (p) fractions were analyzed by SDS-PAGE
and Coomassie blue stain. Quantitation of eEF3 binding was done by densitometric
determination of band intensity and setting the eEF3 binding in the presence of ADPNP to
100% while normalizing according to the intensity of ribosomal protein. Note, that maximum
eEF3 binding is only observed in the presence of the nonhydrolyzable ATP analog ADPNP.
The presence of the antibiotic neomycin stabilizes eEF3 binding also in the presence of ADP,
however, not to levels observed with ADPNP.
b, Fourier shell correlation (FSC) curve for the cryo-EM reconstruction of the yeast RNCeEF3 complex in the presence of ADPNP and neomycin. The resolution is 9.9 Å according to
a cut-off of the FSC at 0.5 (6.2 Å at 3).
Suppl. Figure 6. Complete eEF3-RNC-Sec61 density and difference map.
a, The complete eEF3-RNC-Sec61 density is shown with extra density appearing for eEF3,
which connects the head of the 40S subunit with the central protuberance of the 60S subunit.
Note the additional density at the bottom of the 60S subunit which represents partial
occupation of the exit site with Sec61. Since RNCs carrying a signal sequence were used for
structure determination we added Sec61 to avoid signal sequence induced bias of particle
orientation on the EM grid. b, The same map as in (a) is shown as wire mesh together with a
difference map (red) at 3.5σ (top) and at 2.5σ (middle), comparing the eEF3-RNC-Sec61
density with a RNC-Sec61 map. Bottom: the difference map (red) is shown at 2.5σ together
with the RNC-Sec61 map (blue surface). This difference map together with the RNC-Sec61
map allow very precise assignment of the eEF3 density and its boundaries.
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