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- EMDB-3048: Structure of a partial yeast 48S preinitiation complex in closed ... -

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Entry
Database: EMDB / ID: EMD-3048
TitleStructure of a partial yeast 48S preinitiation complex in closed conformation
Map dataTo see a continuous density for factors eIF1, eIF2, mRNA, entire tRNA and eIF3 at the 40S subunit interface, apply a Gaussian filtering of 1.34 and a contour level of 0.025. To see a continuous density for the PCI domain of eIF3, apply a Gaussian filtering of 2.5 and a contour level of 0.008.
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
  • Protein or peptide: Eukaryotic initiation factor 3
  • 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 / eukaryotic translation initiation factor 3 complex, eIF3e / ABC-family proteins mediated transport / eukaryotic translation initiation factor 3 complex, eIF3m / methionyl-initiator methionine tRNA binding / incipient cellular bud site / translation reinitiation / eukaryotic translation initiation factor 2 complex ...formation of translation initiation ternary complex / Recycling of eIF2:GDP / Cellular response to mitochondrial stress / eukaryotic translation initiation factor 3 complex, eIF3e / ABC-family proteins mediated transport / eukaryotic translation initiation factor 3 complex, eIF3m / methionyl-initiator methionine tRNA binding / incipient cellular bud site / translation reinitiation / eukaryotic translation initiation factor 2 complex / eukaryotic translation initiation factor 3 complex / formation of cytoplasmic translation initiation complex / multi-eIF complex / cytoplasmic translational initiation / protein-synthesizing GTPase / eukaryotic 43S preinitiation complex / formation of translation preinitiation complex / eukaryotic 48S preinitiation complex / positive regulation of translational fidelity / 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 / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / translation regulator activity / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translation initiation factor binding / translation initiation factor activity / rescue of stalled ribosome / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / translational initiation / protein kinase C binding / maintenance of translational fidelity / modification-dependent protein catabolic process / cytoplasmic stress granule / protein tag activity / rRNA processing / ribosomal small subunit biogenesis / double-stranded RNA binding / small ribosomal subunit rRNA binding / ribosome binding / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / ribosome / structural constituent of ribosome / protein ubiquitination / translation / ribonucleoprotein complex / positive regulation of protein phosphorylation / GTPase activity / mRNA binding / ubiquitin protein ligase binding / nucleolus / GTP binding / protein kinase binding / RNA binding / zinc ion binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Eukaryotic translation initiation factor 3 subunit G / Eukaryotic translation initiation factor 3 subunit G, N-terminal / eIF3G, RNA recognition motif / Eukaryotic translation initiation factor 3 subunit G / Eukaryotic translation initiation factor SUI1 / Eukaryotic translation initiation factor 3 subunit I / Eukaryotic translation initiation factor 3 subunit B / eIF3B, RNA recognition motif / Translation initiation factor, beta propellor-like domain / Eukaryotic translation initiation factor eIF2A ...Eukaryotic translation initiation factor 3 subunit G / Eukaryotic translation initiation factor 3 subunit G, N-terminal / eIF3G, RNA recognition motif / Eukaryotic translation initiation factor 3 subunit G / Eukaryotic translation initiation factor SUI1 / Eukaryotic translation initiation factor 3 subunit I / Eukaryotic translation initiation factor 3 subunit B / eIF3B, RNA recognition motif / Translation initiation factor, beta propellor-like domain / Eukaryotic translation initiation factor eIF2A / SUI1 domain superfamily / Translation initiation factor SUI1 / Translation initiation factor SUI1 family profile. / SUI1 domain / eIF3a, PCI domain, TPR-like region / Eukaryotic translation initiation factor 3 subunit A / Eukaryotic translation initiation factor 3 subunit C, N-terminal domain / Eukaryotic translation initiation factor 3 subunit C / Eukaryotic translation initiation factor 3 subunit 8 N-terminus / 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. / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain 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 S26e / Ribosomal protein S21e, conserved site / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein S21e signature. / : / Ribosomal protein S12e signature. / Ribosomal protein S12e / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein S2, eukaryotic / Small (40S) ribosomal subunit Asc1/RACK1 / S27a-like superfamily / 40S Ribosomal protein S10 / Ribosomal protein S10, eukaryotic/archaeal / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S2, eukaryotic/archaeal / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S17e, conserved site / Ribosomal protein S17e signature. / Ribosomal protein S30 / : / Ribosomal protein S30 / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal protein S7e signature. / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein S19e / Translation elongation factor EFTu-like, domain 2 / Ribosomal protein S3Ae, conserved site / Ribosomal protein S19e / Ribosomal protein S3Ae signature. / Ribosomal_S19e / Ribosomal protein S27e signature. / 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
Similarity search - Domain/homology
Eukaryotic translation initiation factor 3 subunit G / KLLA0B11231p / Eukaryotic translation initiation factor 3 subunit B / 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 3 subunit C / Eukaryotic translation initiation factor eIF-1 ...Eukaryotic translation initiation factor 3 subunit G / KLLA0B11231p / Eukaryotic translation initiation factor 3 subunit B / 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 3 subunit C / Eukaryotic translation initiation factor eIF-1 / Small ribosomal subunit protein eS28 / Eukaryotic translation initiation factor 3 subunit A / Eukaryotic translation initiation factor 1A / Eukaryotic translation initiation factor 3 subunit I / Ubiquitin-ribosomal protein eS31 fusion protein / KLLA0F25542p / KLLA0F18040p / KLLA0F09812p / KLLA0F07843p / 40S ribosomal protein S12 / Small ribosomal subunit protein eS6 / KLLA0E23673p / 40S ribosomal protein S8 / Small ribosomal subunit protein uS2 / KLLA0E12277p / 40S ribosomal protein S27 / Small ribosomal subunit protein uS14 / KLLA0D10659p / 40S ribosomal protein S3 / 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 / KLLA0A10483p / KLLA0A07194p / 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: 4.9 Å
AuthorsLlacer JL / Hussain T / Ramakrishnan V
CitationJournal: Mol Cell / Year: 2015
Title: Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex.
Authors: Jose L Llácer / Tanweer Hussain / Laura Marler / Colin Echeverría Aitken / Anil Thakur / Jon R Lorsch / Alan G Hinnebusch / V Ramakrishnan /
Abstract: Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and ...Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.
History
DepositionJun 15, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseAug 5, 2015-
UpdateAug 19, 2015-
Current statusAug 19, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jap
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3048.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTo see a continuous density for factors eIF1, eIF2, mRNA, entire tRNA and eIF3 at the 40S subunit interface, apply a Gaussian filtering of 1.34 and a contour level of 0.025. To see a continuous density for the PCI domain of eIF3, apply a Gaussian filtering of 2.5 and a contour level of 0.008.
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
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.08
Minimum - Maximum-0.23441811 - 0.44270736
Average (Standard dev.)0.00030164 (±0.02307597)
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-0.2340.4430.000

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Supplemental data

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Supplemental map: emd 3048 half map 1.map

Fileemd_3048_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Supplemental map: emd 3048 half map 2.map

Fileemd_3048_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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
  • Protein or peptide: Eukaryotic initiation factor 3
  • 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: 7
Molecular weightTheoretical: 1.75 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: Eukaryotic initiation factor 3

MacromoleculeName: Eukaryotic initiation factor 3 / type: protein_or_peptide / ID: 4 / Name.synonym: eIF3
Details: Uniprot codes are: 3a-P38249, 3b-P06103, 3c-P32497, 3g-A6ZZ25, 3i-P40217
Number of copies: 1 / Oligomeric state: Five subunits, 3a, 3b, 3c, 3g, 3i / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 395 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: GP3511

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Macromolecule #5: Initiator transfer RNA

MacromoleculeName: Initiator transfer RNA / type: rna / ID: 5 / 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 #6: Messenger RNA

MacromoleculeName: Messenger RNA / type: rna / ID: 6 / 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 2.5-3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DetailsComplete dataset was collected in 4 non-consecutive sessions
DateJul 4, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 5500 / Average electron dose: 27 e/Å2
Details: Complete dataset was collected in 4 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: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsParticles were automatically picked with the autopicking tool of Relion
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 21401
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

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

PDB-3jap:
Structure of a partial yeast 48S preinitiation complex in closed conformation

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Atomic model buiding 2

Initial modelPDB ID:

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

PDB-3jap:
Structure of a partial yeast 48S preinitiation complex in closed conformation

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Atomic model buiding 3

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C
SoftwareName: Chimera, Coot, Refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3jap:
Structure of a partial yeast 48S preinitiation complex in closed conformation

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Atomic model buiding 4

Initial modelPDB ID:

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

PDB-3jap:
Structure of a partial yeast 48S preinitiation complex in closed conformation

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Atomic model buiding 5

Initial modelPDB ID:

Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: I / Chain - #2 - Chain ID: G
SoftwareName: Chimera, Coot, Refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3jap:
Structure of a partial yeast 48S preinitiation complex in closed conformation

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