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- PDB-8rw1: Structure of a yeast 48S-AUC preinitiation complex in closed conf... -

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Basic information

Entry
Database: PDB / ID: 8rw1
TitleStructure of a yeast 48S-AUC preinitiation complex in closed conformation
Components
  • (40S ribosomal protein ...) x 12
  • (Eukaryotic translation initiation factor 2 subunit ...) x 3
  • (Small ribosomal subunit protein ...) x 11
  • 18S ribosomal RNA
  • Eukaryotic translation initiation factor 1A
  • KLLA0A07194p
  • KLLA0A10483p
  • KLLA0B01474p
  • KLLA0B01562p
  • KLLA0B08173p
  • KLLA0B11231p
  • KLLA0D10659p
  • KLLA0E12277p
  • KLLA0E23673p
  • KLLA0F07843p
  • KLLA0F18040p
  • Met-tRNAi
  • mRNA (5'-R(P*AP*AP*U)-3')
KeywordsRIBOSOME / translation / initiation factors / 40S / eIF1A / AUC codon / eIF2 / tRNAi / 48S PIC / 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 / protein-synthesizing GTPase ...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 / 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) / 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 / positive regulation of apoptotic signaling pathway / 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 / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / ribonucleoprotein complex / positive regulation of protein phosphorylation / translation / GTPase activity / mRNA binding / ubiquitin protein ligase binding / GTP binding / nucleolus / protein kinase binding / RNA binding / zinc ion binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
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 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 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 / Ribosomal protein S8e signature. / Ribosomal protein S19A/S15e / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal S17 / : / Ribosomal protein S6, eukaryotic / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S4e, N-terminal / RS4NT (NUC023) domain / 40S ribosomal protein S11, N-terminal / Ribosomal_S17 N-terminal / Ribosomal protein S7e / Ribosomal protein S7e / Ribosomal protein S4, KOW domain / Ribosomal protein S4e / Ribosomal protein S4e, central region / Ribosomal protein S4e, central domain superfamily / Ribosomal family S4e / Ribosomal protein S6/S6e/A/B/2, conserved site / Ribosomal protein S6e signature. / Ribosomal protein S23, eukaryotic/archaeal / Ribosomal protein S24e
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / METHIONINE / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / KLLA0B11231p / Eukaryotic translation initiation factor 2 subunit beta / Eukaryotic translation initiation factor 2 subunit alpha / Small ribosomal subunit protein uS11 ...PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / METHIONINE / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / KLLA0B11231p / Eukaryotic translation initiation factor 2 subunit beta / Eukaryotic translation initiation factor 2 subunit alpha / Small ribosomal subunit protein uS11 / Eukaryotic translation initiation factor 2 subunit gamma / Small ribosomal subunit protein eS28 / Eukaryotic translation initiation factor 1A / 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 speciesSaccharomyces cerevisiae S288C (yeast)
Kluyveromyces lactis NRRL Y-1140 (yeast)
Kluyveromyces lactis (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsVillamayor-Belinchon, L. / Sharma, P. / Llacer, J.L. / Hussain, T.
Funding support Spain, India, 2items
OrganizationGrant numberCountry
Ministerio de Ciencia e Innovacion (MCIN)PID2020-116880GB-I00 Spain
Department of Biotechnology (DBT, India)IA/I/17/2/503313 India
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural basis of AUC codon discrimination during translation initiation in yeast.
Authors: Laura Villamayor-Belinchón / Prafful Sharma / Yuliya Gordiyenko / Jose L Llácer / Tanweer Hussain /
Abstract: In eukaryotic translation initiation, the 48S preinitiation complex (PIC) scans the 5' untranslated region of mRNAs to search for the cognate start codon (AUG) with assistance from various ...In eukaryotic translation initiation, the 48S preinitiation complex (PIC) scans the 5' untranslated region of mRNAs to search for the cognate start codon (AUG) with assistance from various eukaryotic initiation factors (eIFs). Cognate start codon recognition is precise, rejecting near-cognate codons with a single base difference. However, the structural basis of discrimination of near-cognate start codons was not known. We have captured multiple yeast 48S PICs with a near-cognate AUC codon at the P-site, revealing that the AUC codon induces instability in the codon-anticodon at the P-site, leading to a disordered N-terminal tail of eIF1A. Following eIF1 dissociation, the N-terminal domain of eIF5 fails to occupy the vacant eIF1 position, and eIF2β becomes flexible. Consequently, 48S with an AUC codon is less favourable for initiation. Furthermore, we observe hitherto unreported metastable states of the eIF2-GTP-Met-tRNAMet ternary complex, where the eIF2β helix-turn-helix domain may facilitate eIF5 association by preventing eIF1 rebinding to 48S PIC. Finally, a swivelled head conformation of 48S PIC appears crucial for discriminating incorrect and selection of the correct codon-anticodon pair during translation initiation.
History
DepositionFeb 2, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
2: 18S ribosomal RNA
A: Small ribosomal subunit protein uS2
B: Small ribosomal subunit protein eS1
C: Small ribosomal subunit protein uS5
E: 40S ribosomal protein S4
G: Small ribosomal subunit protein eS6
H: 40S ribosomal protein S7
I: 40S ribosomal protein S8
J: KLLA0E23673p
L: KLLA0A10483p
N: KLLA0F18040p
O: Small ribosomal subunit protein uS11
V: 40S ribosomal protein S21
W: Small ribosomal subunit protein uS8
X: KLLA0B11231p
Y: 40S ribosomal protein S24
a: 40S ribosomal protein S26
b: 40S ribosomal protein S27
e: 40S ribosomal protein S30
h: 40S ribosomal protein L41-A
D: 40S ribosomal protein S3
F: KLLA0D10659p
K: KLLA0B08173p
M: 40S ribosomal protein S12
P: KLLA0F07843p
Q: Small ribosomal subunit protein uS9
R: KLLA0B01474p
S: KLLA0B01562p
T: KLLA0A07194p
U: Small ribosomal subunit protein uS10
Z: 40S ribosomal protein S25
c: Small ribosomal subunit protein eS28
d: Small ribosomal subunit protein uS14
f: Small ribosomal subunit protein eS31
g: KLLA0E12277p
i: Eukaryotic translation initiation factor 1A
3: mRNA (5'-R(P*AP*AP*U)-3')
1: Met-tRNAi
j: Eukaryotic translation initiation factor 2 subunit alpha
k: Eukaryotic translation initiation factor 2 subunit gamma
l: Eukaryotic translation initiation factor 2 subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,362,123161
Polymers1,358,46141
Non-polymers3,662120
Water543
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area238160 Å2
ΔGint-2536 kcal/mol
Surface area445350 Å2
MethodPISA

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Components

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RNA chain , 3 types, 3 molecules 231

#1: RNA chain 18S ribosomal RNA


Mass: 579454.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast)
#37: RNA chain mRNA (5'-R(P*AP*AP*U)-3')


Mass: 15304.940 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Kluyveromyces lactis (yeast)
#38: RNA chain Met-tRNAi


Mass: 24713.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast)

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Small ribosomal subunit protein ... , 11 types, 11 molecules ABCGOWQUcdf

#2: Protein Small ribosomal subunit protein uS2 / 40S ribosomal protein S0


Mass: 28264.525 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / Strain: ATCC 8585 / References: UniProt: Q6CN12
#3: Protein Small ribosomal subunit protein eS1 / 40S ribosomal protein S1


Mass: 28971.643 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CWD0
#4: Protein Small ribosomal subunit protein uS5 / 40S ribosomal protein S2


Mass: 27649.979 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CKL3
#6: Protein Small ribosomal subunit protein eS6 / 40S ribosomal protein S6


Mass: 26970.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CM04
#12: Protein Small ribosomal subunit protein uS11 / 40S ribosomal protein S14 / RP59


Mass: 14530.655 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: P27069
#14: Protein Small ribosomal subunit protein uS8 / 40S ribosomal protein S22


Mass: 14645.041 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CW21
#26: Protein Small ribosomal subunit protein uS9 / 40S ribosomal protein S16


Mass: 15874.531 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q875N2
#30: Protein Small ribosomal subunit protein uS10 / 40S ribosomal protein S20


Mass: 13337.604 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CIM1
#32: Protein Small ribosomal subunit protein eS28 / 40S ribosomal protein S28 / S33


Mass: 7549.824 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: P33285
#33: Protein Small ribosomal subunit protein uS14 / 40S ribosomal protein S29


Mass: 6662.570 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CPG3
#34: Protein Small ribosomal subunit protein eS31 / 40S ribosomal protein S27a


Mass: 17110.977 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: P69061

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40S ribosomal protein ... , 12 types, 12 molecules EHIVYabehDMZ

#5: Protein 40S ribosomal protein S4


Mass: 29617.514 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CWJ2
#7: Protein 40S ribosomal protein S7


Mass: 21735.297 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CTD6
#8: Protein 40S ribosomal protein S8


Mass: 22642.727 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CMG3
#13: Protein 40S ribosomal protein S21


Mass: 9797.949 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CXT6
#16: Protein 40S ribosomal protein S24


Mass: 15194.549 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CU44
#17: Protein 40S ribosomal protein S26


Mass: 13539.957 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CS01
#18: Protein 40S ribosomal protein S27


Mass: 8884.362 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CNL2
#19: Protein 40S ribosomal protein S30


Mass: 7141.421 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CUH5
#20: Protein/peptide 40S ribosomal protein L41-A


Mass: 3354.243 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast)
#21: Protein 40S ribosomal protein S3


Mass: 26300.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CRK7
#24: Protein 40S ribosomal protein S12


Mass: 14466.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CLU4
#31: Protein 40S ribosomal protein S25


Mass: 12002.116 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CW78

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Protein , 12 types, 12 molecules JLNXFKPRSTgi

#9: Protein KLLA0E23673p


Mass: 21587.049 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CM18
#10: Protein KLLA0A10483p


Mass: 17843.930 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CX80
#11: Protein KLLA0F18040p


Mass: 16989.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CJK0
#15: Protein KLLA0B11231p


Mass: 16047.897 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: F2Z602
#22: Protein KLLA0D10659p


Mass: 25385.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CRA3
#23: Protein KLLA0B08173p


Mass: 12584.377 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CVZ5
#25: Protein KLLA0F07843p


Mass: 15986.796 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CKV4
#27: Protein KLLA0B01474p


Mass: 15722.216 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CWU3
#28: Protein KLLA0B01562p


Mass: 17084.602 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CWT9
#29: Protein KLLA0A07194p


Mass: 15879.010 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CXM0
#35: Protein KLLA0E12277p


Mass: 35830.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Kluyveromyces lactis NRRL Y-1140 (yeast) / References: UniProt: Q6CNI7
#36: Protein Eukaryotic translation initiation factor 1A / eIF-1A / Eukaryotic translation initiation factor 4C / eIF-4C


Mass: 17462.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: TIF11, YMR260C, YM8156.02C / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P38912

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Eukaryotic translation initiation factor 2 subunit ... , 3 types, 3 molecules jkl

#39: Protein Eukaryotic translation initiation factor 2 subunit alpha / eIF-2-alpha / eIF-2A / eIF-2alpha / eIF2-alpha


Mass: 34763.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SUI2, TIF211, YJR007W, J1429 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P20459
#40: Protein Eukaryotic translation initiation factor 2 subunit gamma / eIF2-gamma


Mass: 57942.699 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: GCD11, SUI4, TIF213, YER025W / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P32481, protein-synthesizing GTPase
#41: Protein Eukaryotic translation initiation factor 2 subunit beta / eIF2-beta


Mass: 31631.309 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SUI3, TIF212, YPL237W / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P09064

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Non-polymers , 5 types, 123 molecules

#42: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 115 / Source method: obtained synthetically / Formula: Mg
#43: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#44: Chemical ChemComp-GCP / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GMP-PCP, energy-carrying molecule analogue*YM
#45: Chemical ChemComp-MET / METHIONINE


Type: L-peptide linking / Mass: 149.211 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H11NO2S
#46: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Structure of a yeast 48S-AUC preinitiation complex in closed conformationRIBOSOME#1-#16, #18-#410MULTIPLE SOURCES
2RibosomeCOMPLEX#1-#16, #18-#351NATURAL
3tRNACOMPLEX#381NATURAL
4Initiation factors eIF1ACOMPLEX#361RECOMBINANT
5Initiation factor eIF2COMPLEX#39-#411RECOMBINANT
6mRNACOMPLEX#371RECOMBINANT
Molecular weightValue: 1.8 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37) (yeast)284590
33Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
44Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
55Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
66Kluyveromyces lactis (yeast)28985
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
44Escherichia coli BL21(DE3) (bacteria)469008
55Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
66synthetic construct (others)32630
Buffer solutionpH: 6.5
Buffer component
IDConc.NameBuffer-ID
120 mMMES1
25 mMMagnessium acetate1
380 mMPotassium acetate1
410 mMAmmonium acetate1
52 mMDithiothreitol (DTT)1
60.001 mMZinc acetate1
70.6 mMATP1
80.25 mMGDPCP1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 90 K
Image recordingAverage exposure time: 1.1 sec. / Electron dose: 30 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 8 / Num. of real images: 11245
Details: Images were collected in movie-mode at 30 frames per second

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Processing

EM software
IDNameVersionCategory
1EMANparticle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimera1.1model fitting
8Coot0.8model fitting
10RELION1.4initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
33REFMAC5.8model refinement
34PHENIX1.2model refinement
Image processingDetails: FEI Falcon III
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1680000 / Details: Selected particles after 2d-classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66519 / Symmetry type: POINT
Atomic model buildingB value: 123 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: FSC
Atomic model buildingPDB-ID: 6FYX

6fyx


Accession code: 6FYX / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00391400
ELECTRON MICROSCOPYf_angle_d0.68132604
ELECTRON MICROSCOPYf_dihedral_angle_d18.00228936
ELECTRON MICROSCOPYf_chiral_restr0.03916499
ELECTRON MICROSCOPYf_plane_restr0.0069856

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