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- PDB-7nwt: Initiated 70S ribosome in complex with 2A protein from encephalom... -

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Basic information

Entry
Database: PDB / ID: 7nwt
TitleInitiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV)
Components
  • (30S ribosomal protein ...) x 20
  • (50S ribosomal protein ...) x 31
  • 16S ribosomal RNA
  • 23S ribosomal RNA
  • 5S ribosomal RNA
  • Protein 2A
  • fMet-NH-tRNA(fMet)
  • mRNA
KeywordsRIBOSOME / 70S ribosome / initiation complex / EMCV / cardiovirus / 2A / picornavirus / frameshifting / PRF / RNA-binding protein / protein-mediated frameshifting / ribosome-binding protein / beta-shell / viral protein
Function / homology
Function and homology information


positive stranded viral RNA replication / host cell nucleolus / stringent response / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity ...positive stranded viral RNA replication / host cell nucleolus / stringent response / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / picornain 3C / positive regulation of RNA splicing / cytosolic ribosome assembly / T=pseudo3 icosahedral viral capsid / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / host cell cytoplasmic vesicle membrane / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / symbiont-mediated suppression of host gene expression / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / large ribosomal subunit / channel activity / ribosome biogenesis / ribosome binding / ribosomal small subunit assembly / large ribosomal subunit rRNA binding / 5S rRNA binding / transferase activity / small ribosomal subunit / cytosolic small ribosomal subunit / ribosomal large subunit assembly / monoatomic ion transmembrane transport / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / RNA helicase activity / negative regulation of translation / rRNA binding / ribosome / RNA helicase / structural constituent of ribosome / induction by virus of host autophagy / translation / symbiont entry into host cell / ribonucleoprotein complex / RNA-directed RNA polymerase / viral translational frameshifting / cysteine-type endopeptidase activity / response to antibiotic / RNA-dependent RNA polymerase activity / negative regulation of DNA-templated transcription / mRNA binding / virus-mediated perturbation of host defense response / DNA-templated transcription / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / DNA binding / RNA binding / zinc ion binding / ATP binding / membrane / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Leader peptide, picornavirus / Viral leader polypeptide zinc finger / Virion protein N terminal domain / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 ...Leader peptide, picornavirus / Viral leader polypeptide zinc finger / Virion protein N terminal domain / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / Picornavirus coat protein / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L11, bacterial-type / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / : / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L17 signature. / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal L25p family / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Ribosomal protein L25 / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L32p, bacterial type / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / : / : / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L6, bacterial-type / Picornavirus/Calicivirus coat protein / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein L20 signature. / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein L27, conserved site
Similarity search - Domain/homology
N-FORMYLMETHIONINE / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / 50S ribosomal protein L10 / Small ribosomal subunit protein bS18 ...N-FORMYLMETHIONINE / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / 50S ribosomal protein L10 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS21 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS12 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Genome polyprotein / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25 / Large ribosomal subunit protein bL31
Similarity search - Component
Biological speciesMengo encephalomyocarditis virus
Escherichia coli (E. coli)
Encephalomyocarditis virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsHill, C.H. / Napthine, S. / Pekarek, L. / Kibe, A. / Firth, A.E. / Graham, S.C. / Caliskan, N. / Brierley, I.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Wellcome Trust202797/Z/16/Z United Kingdom
Wellcome Trust098406/Z/12/B United Kingdom
European Communitys Seventh Framework ProgrammeBIOSTRUCT-X (Contract No. 283570)European Union
CitationJournal: Nat Commun / Year: 2021
Title: Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.
Authors: Chris H Hill / Lukas Pekarek / Sawsan Napthine / Anuja Kibe / Andrew E Firth / Stephen C Graham / Neva Caliskan / Ian Brierley /
Abstract: Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we ...Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
History
DepositionMar 17, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 15, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
B: 50S ribosomal protein L2
C: 50S ribosomal protein L3
D: 50S ribosomal protein L4
E: 50S ribosomal protein L5
F: 50S ribosomal protein L6
G: 50S ribosomal protein L9
H: 50S ribosomal protein L10
I: 50S ribosomal protein L11
J: 50S ribosomal protein L13
K: 50S ribosomal protein L14
L: 50S ribosomal protein L15
M: 50S ribosomal protein L16
N: 50S ribosomal protein L17
O: 50S ribosomal protein L18
P: 50S ribosomal protein L19
Q: 50S ribosomal protein L20
R: 50S ribosomal protein L21
S: 50S ribosomal protein L22
T: 50S ribosomal protein L23
U: 50S ribosomal protein L24
V: 50S ribosomal protein L25
W: 50S ribosomal protein L27
X: 50S ribosomal protein L28
Y: 50S ribosomal protein L29
Z: 50S ribosomal protein L30
1: 23S ribosomal RNA
2: 16S ribosomal RNA
3: 5S ribosomal RNA
5: fMet-NH-tRNA(fMet)
a: 50S ribosomal protein L31
b: 50S ribosomal protein L32
c: 50S ribosomal protein L33
d: 50S ribosomal protein L34
e: 50S ribosomal protein L35
f: 50S ribosomal protein L36
g: 30S ribosomal protein S2
h: 30S ribosomal protein S3
i: 30S ribosomal protein S4
j: 30S ribosomal protein S5
k: 30S ribosomal protein S6
l: 30S ribosomal protein S7
m: 30S ribosomal protein S8
n: 30S ribosomal protein S9
o: 30S ribosomal protein S10
p: 30S ribosomal protein S11
q: 30S ribosomal protein S12
r: 30S ribosomal protein S13
s: 30S ribosomal protein S14
t: 30S ribosomal protein S15
u: 30S ribosomal protein S16
v: 30S ribosomal protein S17
w: 30S ribosomal protein S18
x: 30S ribosomal protein S19
y: 30S ribosomal protein S20
z: 30S ribosomal protein S21
XX: mRNA
AA: Protein 2A
BB: Protein 2A
CC: Protein 2A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,297,585499
Polymers2,286,65659
Non-polymers10,929440
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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50S ribosomal protein ... , 31 types, 31 molecules BCDEFGHIJKLMNOPQRSTUVWXYZabcdef

#1: Protein 50S ribosomal protein L2 / ribosomal protein uL2


Mass: 29923.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60422
#2: Protein 50S ribosomal protein L3 / ribosomal protein uL3


Mass: 22277.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60438
#3: Protein 50S ribosomal protein L4 / ribosomal protein uL4


Mass: 22121.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60723
#4: Protein 50S ribosomal protein L5 / ribosomal protein uL5


Mass: 20333.611 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P62399
#5: Protein 50S ribosomal protein L6 / ribosomal protein uL6


Mass: 18932.791 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG55
#6: Protein 50S ribosomal protein L9 / ribosomal protein bL9


Mass: 15789.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7R1
#7: Protein 50S ribosomal protein L10


Mass: 17736.596 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: A0A073UC57
#8: Protein 50S ribosomal protein L11 / ribosomal protein uL11


Mass: 14894.362 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7J7
#9: Protein 50S ribosomal protein L13 / ribosomal protein uL13


Mass: 16050.606 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AA10
#10: Protein 50S ribosomal protein L14 / ribosomal protein uL14


Mass: 13565.067 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADY3
#11: Protein 50S ribosomal protein L15 / ribosomal protein uL15


Mass: 15008.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P02413
#12: Protein 50S ribosomal protein L16 / ribosomal protein uL16


Mass: 15312.269 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADY7
#13: Protein 50S ribosomal protein L17 / ribosomal protein bL17


Mass: 14393.657 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG44
#14: Protein 50S ribosomal protein L18 / ribosomal protein uL18


Mass: 12794.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0C018
#15: Protein 50S ribosomal protein L19 / ribosomal protein bL19


Mass: 13159.278 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7K6
#16: Protein 50S ribosomal protein L20 / ribosomal protein bL20


Mass: 13528.024 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7L3
#17: Protein 50S ribosomal protein L21 / ribosomal protein bL21


Mass: 11586.374 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG48
#18: Protein 50S ribosomal protein L22 / ribosomal protein uL22


Mass: 12253.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P61175
#19: Protein 50S ribosomal protein L23 / ribosomal protein uL23


Mass: 11222.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADZ0
#20: Protein 50S ribosomal protein L24 / ribosomal protein uL24


Mass: 11339.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60624
#21: Protein 50S ribosomal protein L25 / ribosomal protein bL25


Mass: 10713.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P68919
#22: Protein 50S ribosomal protein L27 / ribosomal protein bL27


Mass: 9146.540 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7L8
#23: Protein 50S ribosomal protein L28 / ribosomal protein bL28


Mass: 9027.551 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7M2
#24: Protein 50S ribosomal protein L29 / ribosomal protein uL29


Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7M6
#25: Protein 50S ribosomal protein L30 / ribosomal protein uL30


Mass: 6554.820 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG51
#30: Protein 50S ribosomal protein L31


Mass: 7887.117 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: U9XX47
#31: Protein 50S ribosomal protein L32 / ribosomal protein bL32


Mass: 6463.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7N4
#32: Protein 50S ribosomal protein L33 / ribosomal protein bL33


Mass: 6388.631 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7N9
#33: Protein/peptide 50S ribosomal protein L34 / ribosomal protein bL34


Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7P5
#34: Protein 50S ribosomal protein L35 / Ribosomal protein A / ribosomal protein bL35


Mass: 7313.032 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7Q1
#35: Protein/peptide 50S ribosomal protein L36 / Ribosomal protein B / ribosomal protein bL36


Mass: 4377.390 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7Q6

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RNA chain , 5 types, 5 molecules 1235XX

#26: RNA chain 23S ribosomal RNA


Mass: 941832.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#27: RNA chain 16S ribosomal RNA


Mass: 497405.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 1758835854
#28: RNA chain 5S ribosomal RNA


Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: CP035706.1
#29: RNA chain fMet-NH-tRNA(fMet)


Mass: 24806.922 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 1317722521
#56: RNA chain mRNA


Mass: 37684.395 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: in vitro transcription / Source: (synth.) Encephalomyocarditis virus

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30S ribosomal protein ... , 20 types, 20 molecules ghijklmnopqrstuvwxyz

#36: Protein 30S ribosomal protein S2


Mass: 26781.670 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3TPN2
#37: Protein 30S ribosomal protein S3


Mass: 26031.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SQX2
#38: Protein 30S ribosomal protein S4


Mass: 23514.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SR62
#39: Protein 30S ribosomal protein S5


Mass: 17629.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SR27
#40: Protein 30S ribosomal protein S6 / Small ribosomal subunit protein bS6


Mass: 15727.512 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P02358
#41: Protein 30S ribosomal protein S7 / Small ribosomal subunit protein uS7


Mass: 20055.156 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: K12 / References: UniProt: P02359
#42: Protein 30S ribosomal protein S8


Mass: 14146.557 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SR12
#43: Protein 30S ribosomal protein S9


Mass: 14886.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SRY2
#44: Protein 30S ribosomal protein S10


Mass: 11755.597 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SQT7
#45: Protein 30S ribosomal protein S11


Mass: 13870.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SR57
#46: Protein 30S ribosomal protein S12 / Small ribosomal subunit protein uS12


Mass: 13814.249 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: K12 / References: UniProt: P0A7S3
#47: Protein 30S ribosomal protein S13


Mass: 13128.467 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SR52
#48: Protein 30S ribosomal protein S14


Mass: 11606.560 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SR07
#49: Protein 30S ribosomal protein S15


Mass: 10290.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SSQ7
#50: Protein 30S ribosomal protein S16


Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SYP2
#51: Protein 30S ribosomal protein S17


Mass: 9724.491 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SQY7
#52: Protein 30S ribosomal protein S18


Mass: 9005.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SFP7
#53: Protein 30S ribosomal protein S19


Mass: 10455.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3SQW2
#54: Protein 30S ribosomal protein S20


Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3TRH7
#55: Protein 30S ribosomal protein S21


Mass: 8524.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: C3STZ7

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Protein , 1 types, 3 molecules AABBCC

#57: Protein Protein 2A / P2A / G


Mass: 17831.182 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mengo encephalomyocarditis virus / Production host: Escherichia coli (E. coli) / References: UniProt: P12296

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Non-polymers , 4 types, 442 molecules

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


Mass: 24.305 Da / Num. of mol.: 437 / Source method: obtained synthetically / Formula: Mg
#59: Chemical ChemComp-FME / N-FORMYLMETHIONINE


Type: L-peptide linking / Mass: 177.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H11NO3S
#60: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#61: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV)
Type: RIBOSOME
Details: 70S ribosome subunits and initiator fMet-tRNA purified from E.coli. mRNA template was generated by in vitro transcription EMCV 2A protein was recombinantly expressed in E.coli
Entity ID: #1-#28, #30-#57 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.5
Details: Initiated 70S ribosomes in 50 mM Tris-HCl pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2 were diluted tenfold into 20 mM HEPES pH 7.5, 100 mM potassium acetate, 1.5 mM MgCl2, 2.0 mM DTT. 2A ...Details: Initiated 70S ribosomes in 50 mM Tris-HCl pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2 were diluted tenfold into 20 mM HEPES pH 7.5, 100 mM potassium acetate, 1.5 mM MgCl2, 2.0 mM DTT. 2A protein was dialysed (3K MWCO, 277K, 16 h) into the same buffer. Crosslinking reactions of 50 microliters comprising 75 nM ribosomes, 3.0 micromolar 2A and 2.0 mM bis(sulfosuccinimidyl)suberate (BS3) were performed on ice (30 min) immediately prior to grid preparation.
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2100 mMpotassium acetate1
32.2 mMmagnesium chloride1
42.0 mMdithiothreitol1
55.0 mMTris-HCl1
67.0 mMammonium acetate1
73.0 mMpotassium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 75 nM ribosomes, 3.0 micromolar 2A crosslinked with 2.0 mM bis(sulfosuccinimidyl)suberate (BS3)
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Quantifoil R 2/2 400-mesh copper supports were coated with an additional ~ 60 angstrom layer of amorphous, evaporated carbon by flotation and thoroughly dried before use. Grids were made ...Details: Quantifoil R 2/2 400-mesh copper supports were coated with an additional ~ 60 angstrom layer of amorphous, evaporated carbon by flotation and thoroughly dried before use. Grids were made hydrophilic by glow-discharge in air for 30 s. Three microliters of crosslinking reaction was applied to grids which were then blotted for 4.5 s and vitrified by plunging into liquid ethane using a Vitrobot MK IV (FEI) at 277K, 100% relative humidity.

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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: 75000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.59 sec. / Electron dose: 54.4 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5730
Details: Images were collected as 23-frame movies. Autofocus every 10 micrometres, no drift measurement, 7 sec delay after stage shift and 3 sec delay after image shift

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selection
2EPU2image acquisition
4CTFFIND4CTF correction
5RELION3.1CTF correction
8UCSF Chimera1.15model fittinginitial docking within map
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
15PHENIX1.91.1-4122model refinementrigid body refine per chain
16PHENIX1.91.1-4122model refinementreal space refinement with rotamer, ramachandran and reference model restraints
Image processingDetails: Movie frames were aligned and a dose-weighted average calculated with MotionCor2
CTF correctionDetails: The contrast transfer function (CTF) was estimated per-image using CtfFind4. All further phase+amplitude correction was performed internally in Relion. Per-particle CTF refinement was done after polishing
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 820475
Details: Reference-free autopicking of particles was performed using the Laplacian-of-Gaussian function in Relion (200 - 250 angstrom diameter)
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120749 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5MDZ

5mdz


Accession code: 5MDZ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50.59 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0103162176
ELECTRON MICROSCOPYf_angle_d0.8065241961
ELECTRON MICROSCOPYf_chiral_restr0.04830766
ELECTRON MICROSCOPYf_plane_restr0.005913403
ELECTRON MICROSCOPYf_dihedral_angle_d14.05574964

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