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

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Basic information

Entry
Database: EMDB / ID: EMD-1417
TitleCryo-EM study of the Spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins
Map data
SampleSpinacea oleracea chloroplast 70S ribosome:
ribosome-eukaryote
Function / homology
Function and homology information


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 ...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
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 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
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 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 speciesSpinacia oleracea (spinach)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.4 Å
AuthorsSharma MR / Wilson DN / Datta PP / Barat C / Schluenzen F / Fucini P / Agrawal RK
CitationJournal: 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 ...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 ReportSummary, Full report, XML, About validation report
History
DepositionAug 31, 2007-
Header (metadata) releaseJan 12, 2010-
Map releaseJan 12, 2010-
UpdateOct 10, 2012-
Current statusOct 10, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 30
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v61
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1417.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.76 Å/pix.
x 130 pix.
= 358.8 Å
2.76 Å/pix.
x 130 pix.
= 358.8 Å
2.76 Å/pix.
x 130 pix.
= 358.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.76 Å
Density
Contour LevelPrimary: 49.0 / Movie #1: 30
Minimum - Maximum-126.152000000000001 - 262.374000000000024
Average (Standard dev.)3.28534 (±26.942900000000002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 358.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.762.762.76
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z358.800358.800358.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-30-30-49
NX/NY/NZ6060100
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-126.152262.3743.285

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Supplemental data

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Sample components

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Entire Spinacea oleracea chloroplast 70S ribosome

EntireName: Spinacea oleracea chloroplast 70S ribosome / Number of components: 1
MassTheoretical: 2.5 MDa

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

Ribosome-eukaryoteName: 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 ...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
MassExperimental: 2.5 MDa
SourceSpecies: Spinacia oleracea (spinach)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionBuffer solution: 10mM Tris-HCL pH 7.6, 50mM KCL, 10mM MgOAc, 7mM 2-ME
pH: 7.6
Support filmquantifoil 300 mesh copper grid
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 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 HolderHolder: Cryo Transfer Holder / Model: OTHER / Temperature: 93
CameraDetector: KODAK SO-163 FILM

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Image acquisition

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

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Image processing

ProcessingMethod: 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 reconstructionAlgorithm: Projection matching procedure / Software: SPIDER / CTF correction: each micrograph / Resolution: 9.4 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: 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
Output model

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