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- EMDB-2763: CryoEM structure of a partial yeast 48S preinitiation complex -

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Entry
Database: EMDB / ID: EMD-2763
TitleCryoEM structure of a partial yeast 48S preinitiation complex
Map dataReconstruction of a partial 48S preinitiation complex from yeast. In order to see all the components it is recommended to apply a gaussian filter of 1.5 width and then visualize it at a contour level of 0.02.
Sample
  • Sample: Partial yeast 48S preinitiation complex
  • Complex: Ribosome small subunit
  • Protein or peptide: Eukaryotic initiation factor 1
  • Protein or peptide: Eukaryotic initiation factor 1A
  • Protein or peptide: Eukaryotic initiation factor 2
  • RNA: Initiator transfer RNA
  • RNA: Messenger RNA
KeywordsEukaryotic translation initiation / 48S / small ribosome subunit.
Function / homology
Function and homology information


formation of translation initiation ternary complex / Recycling of eIF2:GDP / Cellular response to mitochondrial stress / ABC-family proteins mediated transport / methionyl-initiator methionine tRNA binding / translation reinitiation / eukaryotic translation initiation factor 2 complex / formation of cytoplasmic translation initiation complex / multi-eIF complex / eukaryotic 43S preinitiation complex ...formation of translation initiation ternary complex / Recycling of eIF2:GDP / Cellular response to mitochondrial stress / ABC-family proteins mediated transport / methionyl-initiator methionine tRNA binding / translation reinitiation / eukaryotic translation initiation factor 2 complex / formation of cytoplasmic translation initiation complex / multi-eIF complex / eukaryotic 43S preinitiation complex / formation of translation preinitiation complex / eukaryotic 48S preinitiation complex / positive regulation of translational fidelity / protein-synthesizing GTPase / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / Formation of a pool of free 40S subunits / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal small subunit binding / 90S preribosome / regulation of translational fidelity / translation regulator activity / translation initiation factor binding / translation initiation factor activity / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / translational initiation / maintenance of translational fidelity / cytoplasmic stress granule / rRNA processing / double-stranded RNA binding / regulation of translation / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / ribosome / translation / ribonucleoprotein complex / GTPase activity / mRNA binding / protein kinase binding / GTP binding / nucleolus / RNA binding / zinc ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Eukaryotic translation initiation factor SUI1 / SUI1 domain superfamily / Translation initiation factor SUI1 / Translation initiation factor SUI1 family profile. / SUI1 domain / Translation initiation factor IF2/IF5 domain / Translation initiation factor IF2/IF5, N-terminal / Translation initiation factor IF2/IF5, zinc-binding / Translation initiation factor IF2/IF5 / Domain found in IF2B/IF5 ...Eukaryotic translation initiation factor SUI1 / SUI1 domain superfamily / Translation initiation factor SUI1 / Translation initiation factor SUI1 family profile. / SUI1 domain / Translation initiation factor IF2/IF5 domain / Translation initiation factor IF2/IF5, N-terminal / Translation initiation factor IF2/IF5, zinc-binding / Translation initiation factor IF2/IF5 / Domain found in IF2B/IF5 / domain present in translation initiation factor eIF2B and eIF5 / Translation initiation factor 1A (eIF-1A), conserved site / Eukaryotic initiation factor 1A signature. / eukaryotic translation initiation factor 1A / Translation initiation factor 1A (eIF-1A) / Translation initiation factor 2, alpha subunit / Translation initiation factor 2, alpha subunit, middle domain superfamily / Translation initiation factor 2, alpha subunit, C-terminal / IF2a, S1-like domain / Eukaryotic translation initiation factor 2 alpha subunit / Initiation factor eIF2 gamma, C-terminal / Initiation factor eIF2 gamma, domain 2 / Initiation factor eIF2 gamma, GTP-binding domain / Initiation factor eIF2 gamma, C terminal / : / RNA-binding domain, S1, IF1 type / Translation initiation factor 1A / IF-1 / S1 domain IF1 type profile. / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / S1 domain profile. / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / : / Ribosomal protein S12e signature. / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein S12e / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein S5, eukaryotic/archaeal / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein S2, eukaryotic / S27a-like superfamily / Translation elongation factor EFTu-like, domain 2 / 40S Ribosomal protein S10 / : / Ribosomal protein S7e signature. / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S10, eukaryotic/archaeal / Ribosomal protein S30 / Ribosomal protein S17e, conserved site / Ribosomal protein S30 / Ribosomal protein S17e signature. / Ribosomal protein S3Ae, conserved site / Ribosomal protein S3Ae signature. / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S2, eukaryotic/archaeal / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal protein S27e signature. / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S4e signature. / 40S ribosomal protein S4, C-terminal domain / 40S ribosomal protein S4 C-terminus / Ribosomal protein S8e, conserved site / Ribosomal protein S8e signature. / Elongation factor Tu domain 2 / Ribosomal protein S19e / Ribosomal protein S19e / Ribosomal_S19e / Ribosomal protein S6, eukaryotic / Ribosomal protein S19A/S15e / Ribosomal protein S7e / Ribosomal protein S7e / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal S17 / 40S ribosomal protein S11, N-terminal / Ribosomal_S17 N-terminal / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / : / Ribosomal protein S4e, N-terminal / RS4NT (NUC023) domain / Ribosomal protein S4, KOW domain
Similarity search - Domain/homology
KLLA0B11231p / Eukaryotic translation initiation factor 2 subunit beta / Small ribosomal subunit protein eS32A / Eukaryotic translation initiation factor 2 subunit alpha / Small ribosomal subunit protein uS11 / Eukaryotic translation initiation factor 2 subunit gamma / Eukaryotic translation initiation factor eIF-1 / Small ribosomal subunit protein eS28 / Eukaryotic translation initiation factor 1A / Ubiquitin-ribosomal protein eS31 fusion protein ...KLLA0B11231p / Eukaryotic translation initiation factor 2 subunit beta / Small ribosomal subunit protein eS32A / Eukaryotic translation initiation factor 2 subunit alpha / Small ribosomal subunit protein uS11 / Eukaryotic translation initiation factor 2 subunit gamma / Eukaryotic translation initiation factor eIF-1 / Small ribosomal subunit protein eS28 / Eukaryotic translation initiation factor 1A / Ubiquitin-ribosomal protein eS31 fusion protein / Small ribosomal subunit protein uS10 / KLLA0F18040p / Small ribosomal subunit protein uS5 / KLLA0F07843p / 40S ribosomal protein S12 / Small ribosomal subunit protein eS6 / RP21 / 40S ribosomal protein S8 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein RACK1 / 40S ribosomal protein S27 / Small ribosomal subunit protein uS14 / KLLA0D10659p / Small ribosomal subunit protein uS3 / 40S ribosomal protein S26 / 40S ribosomal protein S7 / 40S ribosomal protein S24 / 40S ribosomal protein S30 / KLLA0B08173p / Small ribosomal subunit protein uS8 / 40S ribosomal protein S25 / Small ribosomal subunit protein eS1 / 40S ribosomal protein S4 / KLLA0B01562p / KLLA0B01474p / RP41 / S16a / Small ribosomal subunit protein eS21 / Small ribosomal subunit protein uS9
Similarity search - Component
Biological speciesKluyveromyces lactis (yeast) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsHussain T / Llacer JL / Fernandez IS / Savva CG / Ramakrishnan V
CitationJournal: Cell / Year: 2014
Title: Structural changes enable start codon recognition by the eukaryotic translation initiation complex.
Authors: Tanweer Hussain / Jose L Llácer / Israel S Fernández / Antonio Munoz / Pilar Martin-Marcos / Christos G Savva / Jon R Lorsch / Alan G Hinnebusch / V Ramakrishnan /
Abstract: During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the ...During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.
History
DepositionAug 26, 2014-
Header (metadata) releaseOct 1, 2014-
Map releaseNov 5, 2014-
UpdateJul 8, 2015-
Current statusJul 8, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j81
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2763.png

emd_2763.png

Downloads & links

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Map

FileDownload / File: emd_2763.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a partial 48S preinitiation complex from yeast. In order to see all the components it is recommended to apply a gaussian filter of 1.5 width and then visualize it at a contour level of 0.02.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 300 pix.
= 402. Å		1.34 Å/pix.
x 300 pix.
= 402. Å		1.34 Å/pix.
x 300 pix.
= 402. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.16446616 - 0.46707994
Average (Standard dev.)0.00025123 (±0.02283158)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 402.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z402.000402.000402.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1640.4670.000

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Supplemental data

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Supplemental map: emd 2763 additional 1.map

Fileemd_2763_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Partial yeast 48S preinitiation complex

EntireName: Partial yeast 48S preinitiation complex
Components
  • Sample: Partial yeast 48S preinitiation complex
  • Complex: Ribosome small subunit
  • Protein or peptide: Eukaryotic initiation factor 1
  • Protein or peptide: Eukaryotic initiation factor 1A
  • Protein or peptide: Eukaryotic initiation factor 2
  • RNA: Initiator transfer RNA
  • RNA: Messenger RNA

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Supramolecule #1000: Partial yeast 48S preinitiation complex

SupramoleculeName: Partial yeast 48S preinitiation complex / type: sample / ID: 1000 / Oligomeric state: 1 / Number unique components: 6
Molecular weightTheoretical: 1.4 MDa

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Supramolecule #1: Ribosome small subunit

SupramoleculeName: Ribosome small subunit / type: complex / ID: 1 / Name.synonym: 40S / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S, SSU RNA 18S
Source (natural)Organism: Kluyveromyces lactis (yeast)
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: Eukaryotic initiation factor 1

MacromoleculeName: Eukaryotic initiation factor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: eIF1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 12.3 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pTYB2
SequenceUniProtKB: Eukaryotic translation initiation factor eIF-1

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Macromolecule #2: Eukaryotic initiation factor 1A

MacromoleculeName: Eukaryotic initiation factor 1A / type: protein_or_peptide / ID: 2 / Name.synonym: eIF1A / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast
Molecular weightTheoretical: 17.4 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pTYB2
SequenceUniProtKB: Eukaryotic translation initiation factor 1A

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Macromolecule #3: Eukaryotic initiation factor 2

MacromoleculeName: Eukaryotic initiation factor 2 / type: protein_or_peptide / ID: 3 / Name.synonym: eIF2
Details: Uniprot codes are: alpha-P20459 beta-P09064 gamma-P32481
Number of copies: 1 / Oligomeric state: Three subunits, alpha, beta, gamma / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 124 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: GP3511

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Macromolecule #4: Initiator transfer RNA

MacromoleculeName: Initiator transfer RNA / type: rna / ID: 4 / Name.synonym: Met-tRNAi
Details: Doble mutation (G31U:C39A) when compared with yeast WT initiator tRNA.
Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 23 KDa
SequenceString:
AGCGCCGUGG CGCAGUGGAA GCGCGCAGGU CUCAUAAACC UGAUGUCCUC GGAUCGAAAC CGAGCGGCGC UACCA

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Macromolecule #5: Messenger RNA

MacromoleculeName: Messenger RNA / type: rna / ID: 5 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 7.4 KDa
SequenceString:
GGAAUCUCUC UCUAUGCUCU CUCUC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.17 mg/mL
BufferpH: 6.5
Details: 20mM MES-KOH, 40mM K-acetate, 10mM NH4-acetate, 8mM Mg-acetate, 2mM DTT
GridDetails: Quantifoil R2/2 400 mesh copper grids with 4-5 nm thin carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I / Timed resolved state: 30 second incubation time / Method: Blot for 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DetailsComplete dataset was collected in 5 non-consecutive sessions
DateOct 28, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1791 / Average electron dose: 28 e/Å2
Details: Complete dataset was collected in 5 non-consecutive sessions
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 0.004 µm / Nominal defocus min: 0.0016 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsParticles were semi-automatically picked with the e2boxer.py program of EMAN2
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 29698
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3u5b
PDB Unreleased entry


Chain - Chain ID: 2
SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3j81:
CryoEM structure of a partial yeast 48S preinitiation complex

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Atomic model buiding 2

Initial modelPDB ID:

3u5c
PDB Unreleased entry

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3j81:
CryoEM structure of a partial yeast 48S preinitiation complex

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Atomic model buiding 3

Initial modelPDB ID:

3v11

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3j81:
CryoEM structure of a partial yeast 48S preinitiation complex

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