EMDB-1417: Cryo-EM study of the Spinach chloroplast ribosome reveals the str...

Basic information

• Entry: Database: EMDB / ID: EMD-1417
• Title: Cryo-EM study of the Spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins
• Sample: Spinacea oleracea chloroplast 70S ribosome:; ribosome-eukaryote

Function / homology

Function:

plastid small ribosomal subunit / mitochondrial small ribosomal subunit / mitochondrial translation / cytosolic ribosome / mitochondrial large ribosomal subunit / maturation of LSU-rRNA / ribosome assembly / DNA-templated transcription, termination / chloroplast / positive regulation of translational fidelity / ribosomal small subunit assembly / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / mRNA 5'-UTR binding / small ribosomal subunit rRNA binding / large ribosomal subunit rRNA binding / cytoplasmic translation / large ribosomal subunit / cytosolic large ribosomal subunit / ribosomal large subunit assembly / cytosolic small ribosomal subunit / small ribosomal subunit / transferase activity / negative regulation of translation / ribosome / rRNA binding / structural constituent of ribosome / translation / mRNA binding / response to antibiotic / mitochondrion / RNA binding

Domain/homology:

Ribosomal S11, conserved site / Ribosomal protein L33, conserved site / Ribosomal protein L10e/L16 / Ribosomal protein S4, conserved site / Ribosomal protein S2, conserved site / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein L21, conserved site / Ribosomal protein L22/L17, conserved site / Ribosomal protein L35, conserved site / K homology domain-like, alpha/beta / Ribosomal protein S13, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S18, conserved site / Ribosomal protein S3, conserved site / Ribosomal protein L14P, conserved site / Ribosomal protein S13, bacterial-type / Ribosomal protein S11, bacterial-type / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L2, domain 3 / Ribosomal protein S9, conserved site / Zinc-binding ribosomal protein / Ribosomal protein S12/S23 / Ribosomal protein L11, bacterial-type / Translation protein SH3-like domain superfamily / K homology domain superfamily, prokaryotic type / S15/NS1, RNA-binding / Ribosomal protein S13-like, H2TH / Nucleic acid-binding, OB-fold / Ribosomal protein L2, domain 2 / Nucleotide-binding alpha-beta plait domain superfamily / Ribosomal protein L23/L15e core domain superfamily / 50S ribosomal protein uL4 / Ribosomal protein L25/L23 / Ribosomal protein S5, N-terminal / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / Ribosomal protein S16, conserved site / Ribosomal protein L1, bacterial-type / Ribosomal protein L21-like / Ribosomal protein L1, conserved site / Ribosomal protein L1-like / Ribosomal protein S7 domain / Ribosomal protein S16 domain superfamily / 30s ribosomal protein S13, C-terminal / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L5, C-terminal / Ribosomal protein S19, superfamily / Ribosomal protein L5, N-terminal / Ribosomal protein L20, C-terminal / Ribosomal protein S6 superfamily / Ribosomal protein S8 superfamily / L21-like superfamily / Ribosomal protein L22/L17 superfamily / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein L4 domain superfamily / Ribosomal protein S7, conserved site / Ribosomal protein S19 conserved site / Ribosomal protein L11, C-terminal / Ribosomal protein L11, N-terminal / Ribosomal protein L11, conserved site / Ribosomal protein L16, conserved site / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L5, conserved site / Ribosomal protein L5, bacterial-type / Ribosomal protein L35 / Ribosomal protein L13, conserved site / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L2, C-terminal / Ribosomal protein L2, conserved site / Ribosomal protein S4/S9 / Ribosomal protein L5 domain superfamily / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L24/L26, conserved site / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein S5/S7 / Ribosomal protein S18 / Ribosomal protein S3, C-terminal / Ribosomal protein S14 / Ribosomal protein L22/L17 / Ribosomal protein L11/L12 / Ribosomal protein S5 / KOW / Ribosomal protein S8 / Ribosomal protein S15 / Ribosomal protein S6 / Ribosomal protein S16 / Ribosomal protein L34 / Ribosomal protein L14P / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L16 / Ribosomal protein S7 domain superfamily / Ribosomal protein L14 superfamily / Ribosomal protein S18 superfamily / Ribosomal protein L13 superfamily / Ribosomal protein L10e/L16 superfamily / Ribosomal protein S11 superfamily

Component:

50S ribosomal protein L5, chloroplastic / 30S ribosomal protein S16, chloroplastic / 50S ribosomal protein L11, chloroplastic / 30S ribosomal protein S12, chloroplastic / 30S ribosomal protein S7, chloroplastic / 30S ribosomal protein S13, chloroplastic / 50S ribosomal protein L23, chloroplastic / 50S ribosomal protein L34, chloroplastic / 30S ribosomal protein S9, chloroplastic / 30S ribosomal protein S6 alpha, chloroplastic / 50S ribosomal protein L19, chloroplastic / 50S ribosomal protein L1, chloroplastic / 50S ribosomal protein L32, chloroplastic / 30S ribosomal protein S15, chloroplastic / 30S ribosomal protein S18, chloroplastic / 50S ribosomal protein L33, chloroplastic / 30S ribosomal protein S4, chloroplastic / 50S ribosomal protein L20, chloroplastic / 50S ribosomal protein L22, chloroplastic / 50S ribosomal protein L4, chloroplastic / 30S ribosomal protein S11, chloroplastic / 30S ribosomal protein S14, chloroplastic / 30S ribosomal protein S19, chloroplastic / 50S ribosomal protein L2, chloroplastic / 30S ribosomal protein S2, chloroplastic / 30S ribosomal protein S3, chloroplastic / 50S ribosomal protein L24, chloroplastic / 50S ribosomal protein L14, chloroplastic / 30S ribosomal protein S8, chloroplastic / 50S ribosomal protein L13, chloroplastic / 50S ribosomal protein L16, chloroplastic / 50S ribosomal protein L35, chloroplastic / 50S ribosomal protein L21, chloroplastic / 30S ribosomal protein S5, chloroplastic

• Biological species: Spinacia oleracea (spinach)
• Method: single particle reconstruction / cryo EM / Resolution: 9.4 Å
• Authors: Sharma MR / Wilson DN / Datta PP / Barat C / Schluenzen F / Fucini P / Agrawal RK
• Citation: Journal: Proc Natl Acad Sci U S A / Year: 2007; Title: Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.; Authors: Manjuli R Sharma / Daniel N Wilson / Partha P Datta / Chandana Barat / Frank Schluenzen / Paola Fucini / Rajendra K Agrawal / ; Abstract: Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.
• Validation Report: Summary, Full report, XML, About validation report

History

Deposition	Aug 31, 2007	-
Header (metadata) release	Jan 12, 2010	-
Map release	Jan 12, 2010	-
Update	Oct 10, 2012	-
Current status	Oct 10, 2012	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_1417.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 2.76 Å

Density

Contour Level	Primary: 49.0 / Movie #1: 30
Minimum - Maximum	-126.152000000000001 - 262.374000000000024
Average (Standard dev.)	3.28534 (±26.942900000000002)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -65 -65 -65 Dimensions 130 130 130 Spacing 130 130 130
Cell	A=B=C: 358.8 Å α=β=γ: 90 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	2.76	2.76	2.76
M x/y/z	130	130	130
origin x/y/z	0.000	0.000	0.000
length x/y/z	358.800	358.800	358.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-30	-30	-49
NX/NY/NZ	60	60	100
MAP C/R/S	1	2	3
start NC/NR/NS	-65	-65	-65
NC/NR/NS	130	130	130
D min/max/mean	-126.152	262.374	3.285

Supplemental data

Sample components

Entire Spinacea oleracea chloroplast 70S ribosome

• Entire: Name: Spinacea oleracea chloroplast 70S ribosome / Number of components: 1
• Mass: Theoretical: 2.5 MDa

Component #1: ribosome-eukaryote, Spinach Chloroplast 70S Ribosome

• Ribosome-eukaryote: Name: Spinach Chloroplast 70S Ribosome / a.k.a: chloro-ribosome; Details: The SSU 30S has PSRP1,2,3, and 4 identified. LSU 50S did not have PRPL25 and PRPL30 density present. pRRF (plastid ribosome recycling factor)is tightly bound to LSU 50S subunit. One of the two PSRPs on the LSU 50S subunit is identified.; Eukaryote: ALL / Recombinant expression: No
• Mass: Experimental: 2.5 MDa
• Source: Species: Spinacia oleracea (spinach)

Experimental details

Sample preparation

• Specimen: Specimen state: Particle / Method: cryo EM
• Sample solution: Buffer solution: 10mM Tris-HCL pH 7.6, 50mM KCL, 10mM MgOAc, 7mM 2-ME ; pH: 7.6
• Support film: quantifoil 300 mesh copper grid
• Vitrification: Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %; Method: 5 microliters applied to the grid then blotted for 3 seconds with Whatman number 1 filter paper before plunging; Details: Vitrification instrument: Cryo-plunger

Electron microscopy imaging

• Experimental equipment: Model: Tecnai F20 / Image courtesy: FEI Company
• Imaging: Microscope: FEI TECNAI F20
• Electron gun: Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Ų / Illumination mode: FLOOD BEAM
• Lens: Magnification: 50000 X (nominal), 50760 X (calibrated); Astigmatism: objective lens astigmatism was corrected at 250K times magnification; Imaging mode: BRIGHT FIELD / Defocus: 1400 - 4400 nm
• Specimen Holder: Holder: Cryo Transfer Holder / Model: OTHER / Temperature: 93
• Camera: Detector: KODAK SO-163 FILM

Image acquisition

• Image acquisition: Number of digital images: 164 / Scanner: ZEISS SCAI / Sampling size: 14 µm / Bit depth: 12

Image processing

• Processing: Method: single particle reconstruction / Number of class averages: 83 / Number of projections: 86370 ; Details: Initially, 192,133 images were selected using automated particle picking program; Applied symmetry: C₁ (asymmetric)
• 3D reconstruction: Algorithm: Projection matching procedure / Software: SPIDER / CTF correction: each micrograph / Resolution: 9.4 Å / Resolution method: FSC 0.5

Atomic model buiding

Modeling #1

Software: O / Refinement protocol: rigid body
Details: Protocol: Rigid Body. Docking of crystallographic structures in 3D map was performed using program O
Input PDB model: 1DD5

1dd5

Output model

4v61