Figures and data in Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Figures
Tables

6 figures and 4 tables

Figures

Figure 1 with 1 supplement

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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

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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 (FSC_work, red); and between that same model and the reconstruction from the other half of the particles (FSC_test, green), for Pf40S (A) and Pf60S (B).
https://doi.org/10.7554/eLife.03080.004

Figure 2 with 3 supplements

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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

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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

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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

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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

Figure 3

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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

Figure 4

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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

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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

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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

Figure 6

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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
Particles	105,247
Pixel size (Å)	1.34
Defocus range (μm)	0.8–3.8
Voltage (kV)	300
Electron dose (e⁻ Å⁻²)	20

	Pf60S	Pf40S
Model composition
Non-hydrogen atoms	124,509	68,858
Protein residues	6,244	4,106
RNA bases	3,460	1,682
Ligands (Zn²⁺/Mg²⁺/emetine)	5/163/0	1/67/1
Refinement
Resolution used for refinement (Å)	3.1	3.3
Map sharpening B-factor (Å²)	−60.3	−79.9
Average B factor (Å²)	113.1	143.2
Rfactor*	0.2294	0.257
Fourier Shell Correlation†	0.86	0.854
Rms deviations
Bonds (Å)	0.006	0.007
Angles (°)	1.20	1.29
Validation (proteins)
Molprobity score	2.45 (96^th percentile)	2.73 (95^th percentile)
Clashscore, all atoms	3.65 (100^th percentile)	4.23 (100^th percentile)
Good rotamers (%)	90.0	86.0
Ramachandran plot
Favored (%)	90.4	85.4
Outliers (%)	2.4	4.2
Validation (RNA)
Correct sugar puckers (%)	97.3	97.5
Good backbone conformations (%)	71.1	70.0

*

Rfactor = Σ||F_obs| − ||F_calc|/Σ|F_obs|.
†

FSC_overall = Σ(N_shell FSC_shell)/Σ(N_shell), where FSC_shell is the FSC in a given shell, N_shell is the number of ‘structure factors’ in the shell. FSC_shell = Σ(F_model F_EM)/(√(Σ(|F|²_model)) √(Σ(F²_EM)).

Table 2

Ribosomal proteins of the Pf40S subunit

https://doi.org/10.7554/eLife.03080.010

Protein names	Uniprot ID	PlasmoDB ID	Chain ID	Built residues	Extensions compared to human	Total number of residues
eS1	RS3A_PLAF7	PF3D7_0322900	B	24–233	245–262	262
uS2	RSSA_PLAF7	PF3D7_1026800	C	10–204	–	263
uS3	Q8IKH8_PLAF7	PF3D7_1465900	D	4–39; 65–78; 97–193; 207–216	–	221
uS4	Q8I3R0_PLAF7	PF3D7_0520000	E	2–186	–	189
eS4	Q8IIU8_PLAF7	PF3D7_1105400	F	2–258	–	261
uS5	Q8IL02_PLAF7	PF3D7_1447000	G	39–262	–	272
eS6	Q8IDR9_PLAF7	PF3D7_1342000	H	1–160; 170–213	249–306	306
uS7	Q8IBN5_PLAF7	PF3D7_0721600	I	7–118; 128–195	–	195
eS7	Q8IET7_PLAF7	PF3D7_1302800	J	3–190	–	194
uS8	O77395_PLAF7	PF3D7_0316800	K	2–130	–	130
eS8	Q8IM10_PLAF7	PF3D7_1408600	L	5–120; 161–213; 216–218	154–163	218
uS9	Q8IAX5_PLAF7	PF3D7_0813900	M	6–143	–	144
uS10	Q8IK02_PLAF7	PF3D7_1003500	N	21–118	–	118
eS10	Q8IBQ5_PLAF7	PF3D7_0719700	O	11–89	–	137
uS11	Q8I3U6_PLAF7	PF3D7_0516200	P	25–151	–	151
uS12	O97248_PLAF7	PF3D7_0306900	Q	2–145	–	145
eS12	RS12_PLAF7	PF3D7_0307100	R	22–78; 85–100; 111–135	10–16	141
uS13	Q8IIA2_PLAF7	PF3D7_1126200	S	12–139	–	156
uS14	C0H4K8_PLAF7	PF3D7_0705700	T	7–54	–	54
uS15	Q8IDB0_PLAF7	PF3D7_1358800	U	3–151	–	151
uS17	O77381_PLAF7	PF3D7_0317600	V	6–25; 36–161	–	161
eS17	Q8I502_PLAF7	PF3D7_1242700	W	3–83; 97–110	–	137
uS19	C0H5C2_PLAF7	PF3D7_1317800	X	21–95; 103–123	–	145
eS19	Q8IFP2_PLAF7	PF3D7_0422400	Y	15–168	1–19	170
eS21	Q8IHS5_PLAF7	PF3D7_1144000	Z	11–82	–	82
eS24	Q8I3R6_PLAF7	PF3D7_0519400	1	3–122	–	133
eS25	Q8ILN8_PLAF7	PF3D7_1421200	2	35–42; 58–84; 97–102	–	105
eS26	O96258_PLAF7	PF3D7_0217800	3	2–96	–	107
eS27	Q8IEN2_PLAF7	PF3D7_1308300	4	7–82	–	82
eS28	Q8IKL9_PLAF7	PF3D7_1461300	5	2–29; 37–66	–	67
eS30	RS30_PLAF7	PF3D7_0219200	6	6–48	–	58
eS31	Q8IM64_PLAF7	PF3D7_1402500	–	Not built	–	149

Table 3

Ribosomal proteins of the Pf60S subunit

https://doi.org/10.7554/eLife.03080.011

Protein names	Uniprot ID	PlasmoDB ID	Chain ID	Built residues	Extensions compared to human	Total number of residues
uL2	Q8I3T9_PLAF7	PF3D7_0516900	D	2–248	–	260
uL3	Q8IJC6_PLAF7	PF3D7_1027800	E	2–381	–	386
uL4	Q8I431_PLAF7	PF3D7_0507100	F	6–395	373–411	411
uL5	Q8IBQ6_PLAF7	PF3D7_0719600	G	8–51; 64–85; 92–106; 124–166	–	173
uL6	Q8IE85_PLAF7	PF3D7_1323100	H	2–186	–	190
eL6	Q8IDV1_PLAF7	PF3D7_1338200	I	9–151; 158–221	110–118; 139–143; 174–182	221
eL8	Q8ILL2_PLAF7	PF3D7_1424400	J	40–46; 54–131; 147–283	11–24;279–283	283
uL13	Q8IJZ7_PLAF7	PF3D7_1004000	K	1–201	–	202
eL13	Q8IAX6_PLAF7	PF3D7_0814000	L	2–212	134–141; 168–174	215
uL14	Q8IE09_PLAF7	PF3D7_1331800	M	8–139	–	139
eL14	Q8ILE8_PLAF7	PF3D7_1431700	N	5–150	1–18	165
uL15	C6KT23_PLAF7	PF3D7_0618300	O	2–148	–	148
eL15	C0H4A6_PLAF7	PF3D7_0415900	P	2–205	–	205
uL16	Q8ILV2_PLAF7	PF3D7_1414300	Q	2–101; 118–206	–	219
uL18	Q8ILL3_PLAF7	PF3D7_1424100	R	5–126; 141–185; 189–250; 271–293	–	294
eL18	C0H5G3_PLAF7	PF3D7_1341200	U	5–184	–	184
eL19	C6KSY6_PLAF7	PF3D7_0614500	T	2–182	–	182
eL20	Q8IDS6_PLAF7	PF3D7_1341200	S	2–187	–	184
eL21	Q8ILK3_PLAF7	PF3D7_1426000	V	4–158	–	161
uL22	Q8IDI5_PLAF7	PF3D7_1351400	W	4–154; 197–215	–	203
eL22	Q8IB51_PLAF7	PF3D7_0821700	X	40–136	4–18; 34–38	139
uL23	Q8IE82_PLAF7	PF3D7_1323400	Y	88–188	13–34; 57–67	190
uL24	O77364_PLAF7	PF3D7_0312800	Z	2–122	–	126
eL24	Q8IEM3_PLAF7	PF3D7_1309100	0	8–69	–	162
eL27	Q8IKM5_PLAF7	PF3D7_1460700	1	2–126;132–146	–	146
eL28	Q8IHU0_PLAF7	PF3D7_1142500	2	2–69; 77–82; 86–98; 103–119	–	127
uL29	Q8IIB4_PLAF7	PF3D7_1124900	3	3–121	–	124
eL29	C6S3J6_PLAF7	PF3D7_1460300	4	2–67	–	67
uL30	O97250_PLAF7	PF3D7_0307200	5	35–257	–	257
eL30	Q8IJK8_PLAF7	PF3D7_1019400	6	8–105	–	108
eL31	Q8I463_PLAF7	PF3D7_0503800	7	15–88; 95–116	–	120
eL32	Q8I3B0_PLAF7	PF3D7_0903900	8	2–126	–	131
eL33	Q8IHT9_PLAF7	PF3D7_1142600	9	35–137	1–35	140
eL34	Q8IBY4_PLAF7	PF3D7_0710600	a	2–107	–	150
eL36	Q8I713_PLAF7	PF3D7_1109900	b	2–27; 38–106	5–10	112
eL37	C0H4L5_PLAF7	PF3D7_0706400	c	2–90	–	92
eL38	Q8II62_PLAF7	PF3D7_1130100	d	2–31; 36–77	–	87
eL39	C0H4H3_PLAF7	PF3D7_0611700	e	2–30; 38–51	–	51
eL40	Q8ID50_PLAF7	PF3D7_1365900	f	1–51	–	52
eL41	C6S3G4_PLAF7	PF3D7_1144300	g	3–39	1–14	39
eL43	RL37A_PLAF7	PF3D7_0210100.1	h	2–86	–	96
eL44	RL44_PLAF7	PF3D7_0304400	i	2–96	–	104

Table 4

Comparison of ESs in Pf80S and human cytoplasmic ribosomes

https://doi.org/10.7554/eLife.03080.012

rRNA	ES	Helix	Comparison between Pf80S and human ribosomes
18S	ES2S		Shorter loop in Pf80S
	ES3S	A	Conserved
		B	Truncated in Pf80S
	ES13S		Conserved
	ES6S	A	Expanded in Pf80S
		B	Truncated in Pf80S
		C	Conserved
		D	Expanded in Pf80S
		E	Conserved
	ES7S		Expanded in Pf80S
	ES14S		Conserved
	ES9S		Expanded in Pf80S
	ES10S		Expanded in Pf80S
	ES12S		Helix truncated in Pf80S
28S	ES3L		Conserved
	ES4L		Conserved
	ES5L		Conserved
	ES7L	A	Truncated in Pf80S
		B	Truncated. Loop in Pf80S forms a novel interaction with eL14
		B1	Pf-specific ES
		C	Present
		D–H	Absent from Pf80S
	ES8L	H28	Expanded in Pf80S
	ES9L	A	Absent in Pf80S
		H30	Conserved
		H31	Conserved
	ES10L		Absent in Pf80S
	ES12L		Expanded in Pf80S
	ES15L	A	Truncated in Pf80S
	ES19L		Truncated in Pf80S
	ES20L	A	Absent in Pf80S
		B	Conserved in Pf80S
	ES26L		Expanded in Pf80S
	ES27L	A–C	Not present in Pf80S model, predicted divergence between Pf and human cytoplasmic ribosomes
	ES30L		Absent in Pf80S
	ES31L	A	Conserved
		B	Expanded in Pf80S
		C	Conserved
	ES34L		Pf-specific ES
	ES36L		Pf-specific ES
	ES39L	A	Conserved; preceding loop in Pf80S forms a short helix (3 base pairs) with the 5′ end of the 5.8S rRNA
		B	Conserved
	ES41L		Conserved