1. Structural Biology and Molecular Biophysics
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Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

  1. Wilson Wong
  2. Xiao-chen Bai
  3. Alan Brown
  4. Israel S Fernandez
  5. Eric Hanssen
  6. Melanie Condron
  7. Yan Hong Tan
  8. Jake Baum  Is a corresponding author
  9. Sjors HW Scheres  Is a corresponding author
  1. Walter and Eliza Hall Institute of Medical Research, Australia
  2. University of Melbourne, Australia
  3. Medical Research Council Laboratory of Molecular Biology, United Kingdom
  4. Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Australia
Research Article
Cite this article as: eLife 2014;3:e03080 doi: 10.7554/eLife.03080
6 figures, 4 tables and 13 data sets

Figures

Figure 1 with 1 supplement
Cryo-EM data and processing.

(A) Sucrose gradient purification of Pf80S ribosomes. (B) Representative electron micrograph showing Pf80S particles. (C) Fourier Shell Correlation (FSC) curves indicating the overall resolutions of unmasked (red), Pf40S masked (green) and Pf60S masked (blue) reconstructions of the Pf80S–emetine complex. (D) Representative density with built models of a β-strand with well-resolved side chains (left), an RNA segment with separated bases (middle), and a magnesium ion (green sphere) that is coordinated by RNA backbone phosphates. (E) Density maps colored according to local resolution for the unmasked Pf80S (left) and masked Pf40S and Pf60S subunits (right).

https://doi.org/10.7554/eLife.03080.003
Figure 1—figure supplement 1
FSC curves between the final refined atomic model and the reconstructions from all particles (black); between the model refined in the reconstruction from only half of the particles and the reconstruction from that same half (FSCwork, red); and between that same model and the reconstruction from the other half of the particles (FSCtest, green), for Pf40S (A) and Pf60S (B).
https://doi.org/10.7554/eLife.03080.004
Figure 2 with 3 supplements
Structure of the Pf80S ribosome.

Overview of Pf80S atomic model showing views facing (A) tRNA entry side and (B) tRNA exit side. rRNAs are shown in gray, proteins numbered according to Ban et al. (2014). (C and D) Pf40S and Pf60S subunits are colored in yellow and blue respectively. Flexible regions are shown in red and at a resolution of 6 Å. Pf-specific expansion segments (ESs) relative to human ribosomes are labeled. Their numbering is as described for the human cytoplasmic ribosome (Anger et al., 2013).

https://doi.org/10.7554/eLife.03080.005
Figure 2—figure supplement 1
Secondary structure of Pf18S rRNAs.

Pf-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (Cannone et al., 2002).

https://doi.org/10.7554/eLife.03080.006
Figure 2—figure supplement 2
Secondary structure of the 5′ half of Pf 28S rRNA.

Pf-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (Cannone et al., 2002).

https://doi.org/10.7554/eLife.03080.007
Figure 2—figure supplement 3
Secondary structure of the 3′ half of Pf28S rRNA.

Pf-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (Cannone et al., 2002).

https://doi.org/10.7554/eLife.03080.008
Details of Pf-specific protein extensions and rRNA ESs near the (A and B) subunit interface (C) P stalk and (D) the L1 stalk.

Pf-specific elements are shown in red.

https://doi.org/10.7554/eLife.03080.013
Emetine binds to the E-site of the Pf40S subunit.

(A) 2D chemical structure of emetine. (B) A 4.5 Å filtered difference map (red density) at 5 standard deviation overlaid with the Pf80S map filtered at 6 Å (blue and yellow for Pf60S and Pf40S respectively), showing the emetine density at the E-site of the Pf40S. The emetine binding site in (C) empty and (D) emetine-bound structures, with (E) density for emetine alone at 3.2 Å.

https://doi.org/10.7554/eLife.03080.014
Figure 5 with 1 supplement
Molecular details of the emetine–ribosome interaction.

(A) Overview of emetine at the binding interface formed by the three conserved rRNA helices and uS11. h23 is in green, h24 in cyan, h45 in blue, uS11 in pink, and emetine in yellow. (B) 2D representation showing the interaction of emetine with binding residues. Substitution contour represents potential space for chemical modification of emetine. (C) Residues in physical contact with emetine. Hydrogen bond is indicated as dashes.

https://doi.org/10.7554/eLife.03080.015
Figure 5—figure supplement 1
Comparison of the emetine binding residues between Pf80S and human ribosomes.

Human and Pf-specific elements are colored in yellow and cyan respectively, with Pf numbering. Emetine is in purple.

https://doi.org/10.7554/eLife.03080.016
Comparison with pactamycin.

Superposition of emetine and pactamycin at the Pf40S emetine binding pocket. Emetine and pactamycin are shown in yellow and red respectively.

https://doi.org/10.7554/eLife.03080.017

Tables

Table 1

Refinement and model statistics

https://doi.org/10.7554/eLife.03080.009
Pf80S–emetine
Data collection
 Particles105,247
 Pixel size (Å)1.34
 Defocus range (μm)0.8–3.8
 Voltage (kV)300
 Electron dose (e Å−2)20
Pf60SPf40S
Model composition
 Non-hydrogen atoms124,50968,858
 Protein residues6,2444,106
 RNA bases3,4601,682
 Ligands (Zn2+/Mg2+/emetine)5/163/01/67/1
Refinement
 Resolution used for refinement (Å)3.13.3
 Map sharpening B-factor (Å2)−60.3−79.9
 Average B factor (Å2)113.1143.2
 Rfactor*0.22940.257
 Fourier Shell Correlation0.860.854
Rms deviations
 Bonds (Å)0.0060.007
 Angles (°)1.201.29
Validation (proteins)
 Molprobity score2.45 (96th percentile)2.73 (95th percentile)
 Clashscore, all atoms3.65 (100th percentile)4.23 (100th percentile)
 Good rotamers (%)90.086.0
Ramachandran plot
 Favored (%)90.485.4
 Outliers (%)2.44.2
Validation (RNA)
 Correct sugar puckers (%)97.397.5
 Good backbone conformations (%)71.170.0
  1. *

    Rfactor = Σ||Fobs| − ||Fcalc|/Σ|Fobs|.

  2. FSCoverall = Σ(Nshell FSCshell)/Σ(Nshell), where FSCshell is the FSC in a given shell, Nshell is the number of ‘structure factors’ in the shell. FSCshell = Σ(Fmodel FEM)/(√(Σ(|F|2model)) √(Σ(F2EM)).

Table 2

Ribosomal proteins of the Pf40S subunit

https://doi.org/10.7554/eLife.03080.010
Protein namesUniprot IDPlasmoDB IDChain IDBuilt residuesExtensions compared to humanTotal number of residues
eS1RS3A_PLAF7PF3D7_0322900B24–233245–262262
uS2RSSA_PLAF7PF3D7_1026800C10–204263
uS3Q8IKH8_PLAF7PF3D7_1465900D4–39; 65–78; 97–193; 207–216221
uS4Q8I3R0_PLAF7PF3D7_0520000E2–186189
eS4Q8IIU8_PLAF7PF3D7_1105400F2–258261
uS5Q8IL02_PLAF7PF3D7_1447000G39–262272
eS6Q8IDR9_PLAF7PF3D7_1342000H1–160; 170–213249–306306
uS7Q8IBN5_PLAF7PF3D7_0721600I7–118; 128–195195
eS7Q8IET7_PLAF7PF3D7_1302800J3–190194
uS8O77395_PLAF7PF3D7_0316800K2–130130
eS8Q8IM10_PLAF7PF3D7_1408600L5–120; 161–213; 216–218154–163218
uS9Q8IAX5_PLAF7PF3D7_0813900M6–143144
uS10Q8IK02_PLAF7PF3D7_1003500N21–118118
eS10Q8IBQ5_PLAF7PF3D7_0719700O11–89137
uS11Q8I3U6_PLAF7PF3D7_0516200P25–151151
uS12O97248_PLAF7PF3D7_0306900Q2–145145
eS12RS12_PLAF7PF3D7_0307100R22–78; 85–100; 111–13510–16141
uS13Q8IIA2_PLAF7PF3D7_1126200S12–139156
uS14C0H4K8_PLAF7PF3D7_0705700T7–5454
uS15Q8IDB0_PLAF7PF3D7_1358800U3–151151
uS17O77381_PLAF7PF3D7_0317600V6–25; 36–161161
eS17Q8I502_PLAF7PF3D7_1242700W3–83; 97–110137
uS19C0H5C2_PLAF7PF3D7_1317800X21–95; 103–123145
eS19Q8IFP2_PLAF7PF3D7_0422400Y15–1681–19170
eS21Q8IHS5_PLAF7PF3D7_1144000Z11–8282
eS24Q8I3R6_PLAF7PF3D7_051940013–122133
eS25Q8ILN8_PLAF7PF3D7_1421200235–42; 58–84; 97–102105
eS26O96258_PLAF7PF3D7_021780032–96107
eS27Q8IEN2_PLAF7PF3D7_130830047–8282
eS28Q8IKL9_PLAF7PF3D7_146130052–29; 37–6667
eS30RS30_PLAF7PF3D7_021920066–4858
eS31Q8IM64_PLAF7PF3D7_1402500Not built149
Table 3

Ribosomal proteins of the Pf60S subunit

https://doi.org/10.7554/eLife.03080.011
Protein namesUniprot IDPlasmoDB IDChain IDBuilt residuesExtensions compared to humanTotal number of residues
uL2Q8I3T9_PLAF7PF3D7_0516900D2–248260
uL3Q8IJC6_PLAF7PF3D7_1027800E2–381386
uL4Q8I431_PLAF7PF3D7_0507100F6–395373–411411
uL5Q8IBQ6_PLAF7PF3D7_0719600G8–51; 64–85; 92–106; 124–166173
uL6Q8IE85_PLAF7PF3D7_1323100H2–186190
eL6Q8IDV1_PLAF7PF3D7_1338200I9–151; 158–221110–118; 139–143; 174–182221
eL8Q8ILL2_PLAF7PF3D7_1424400J40–46; 54–131; 147–28311–24;279–283283
uL13Q8IJZ7_PLAF7PF3D7_1004000K1–201202
eL13Q8IAX6_PLAF7PF3D7_0814000L2–212134–141; 168–174215
uL14Q8IE09_PLAF7PF3D7_1331800M8–139139
eL14Q8ILE8_PLAF7PF3D7_1431700N5–1501–18165
uL15C6KT23_PLAF7PF3D7_0618300O2–148148
eL15C0H4A6_PLAF7PF3D7_0415900P2–205205
uL16Q8ILV2_PLAF7PF3D7_1414300Q2–101; 118–206219
uL18Q8ILL3_PLAF7PF3D7_1424100R5–126; 141–185; 189–250; 271–293294
eL18C0H5G3_PLAF7PF3D7_1341200U5–184184
eL19C6KSY6_PLAF7PF3D7_0614500T2–182182
eL20Q8IDS6_PLAF7PF3D7_1341200S2–187184
eL21Q8ILK3_PLAF7PF3D7_1426000V4–158161
uL22Q8IDI5_PLAF7PF3D7_1351400W4–154; 197–215203
eL22Q8IB51_PLAF7PF3D7_0821700X40–1364–18; 34–38139
uL23Q8IE82_PLAF7PF3D7_1323400Y88–18813–34; 57–67190
uL24O77364_PLAF7PF3D7_0312800Z2–122126
eL24Q8IEM3_PLAF7PF3D7_130910008–69162
eL27Q8IKM5_PLAF7PF3D7_146070012–126;132–146146
eL28Q8IHU0_PLAF7PF3D7_114250022–69; 77–82; 86–98; 103–119127
uL29Q8IIB4_PLAF7PF3D7_112490033–121124
eL29C6S3J6_PLAF7PF3D7_146030042–6767
uL30O97250_PLAF7PF3D7_0307200535–257257
eL30Q8IJK8_PLAF7PF3D7_101940068–105108
eL31Q8I463_PLAF7PF3D7_0503800715–88; 95–116120
eL32Q8I3B0_PLAF7PF3D7_090390082–126131
eL33Q8IHT9_PLAF7PF3D7_1142600935–1371–35140
eL34Q8IBY4_PLAF7PF3D7_0710600a2–107150
eL36Q8I713_PLAF7PF3D7_1109900b2–27; 38–1065–10112
eL37C0H4L5_PLAF7PF3D7_0706400c2–9092
eL38Q8II62_PLAF7PF3D7_1130100d2–31; 36–7787
eL39C0H4H3_PLAF7PF3D7_0611700e2–30; 38–5151
eL40Q8ID50_PLAF7PF3D7_1365900f1–5152
eL41C6S3G4_PLAF7PF3D7_1144300g3–391–1439
eL43RL37A_PLAF7PF3D7_0210100.1h2–8696
eL44RL44_PLAF7PF3D7_0304400i2–96104
Table 4

Comparison of ESs in Pf80S and human cytoplasmic ribosomes

https://doi.org/10.7554/eLife.03080.012
rRNAESHelixComparison between Pf80S and human ribosomes
18SES2SShorter loop in Pf80S
ES3SAConserved
BTruncated in Pf80S
ES13SConserved
ES6SAExpanded in Pf80S
BTruncated in Pf80S
CConserved
DExpanded in Pf80S
EConserved
ES7SExpanded in Pf80S
ES14SConserved
ES9SExpanded in Pf80S
ES10SExpanded in Pf80S
ES12SHelix truncated in Pf80S
28SES3LConserved
ES4LConserved
ES5LConserved
ES7LATruncated in Pf80S
BTruncated. Loop in Pf80S forms a novel interaction with eL14
B1Pf-specific ES
CPresent
D–HAbsent from Pf80S
ES8LH28Expanded in Pf80S
ES9LAAbsent in Pf80S
H30Conserved
H31Conserved
ES10LAbsent in Pf80S
ES12LExpanded in Pf80S
ES15LATruncated in Pf80S
ES19LTruncated in Pf80S
ES20LAAbsent in Pf80S
BConserved in Pf80S
ES26LExpanded in Pf80S
ES27LA–CNot present in Pf80S model, predicted divergence between Pf and human cytoplasmic ribosomes
ES30LAbsent in Pf80S
ES31LAConserved
BExpanded in Pf80S
CConserved
ES34LPf-specific ES
ES36LPf-specific ES
ES39LAConserved; preceding loop in Pf80S forms a short helix (3 base pairs) with the 5′ end of the 5.8S rRNA
BConserved
ES41LConserved

Data availability

The following data sets were generated
  1. 1
  2. 2
  3. 3
  4. 4
The following previously published data sets were used
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  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9

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