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- PDB-5uz4: The cryo-EM structure of YjeQ bound to the 30S subunit suggests a... -

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Entry
Database: PDB / ID: 5uz4
TitleThe cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly
Components
  • (30S ribosomal protein ...) x 19
  • 16S RIBOSOMAL RNA
  • Small ribosomal subunit biogenesis GTPase RsgA
KeywordsRIBOSOME/Hydrolase / Ribosome assembly / 30S subunit / YjeQ protein / RsgA protein / RIBOSOME-Hydrolase complex
Function/homologyRibosome biogenesis GTPase RsgA / EngC GTPase domain profile. / RsgA GTPase domain / RsgA GTPase / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain profile. / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / ec:3.6.1.-: / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / ribosomal small subunit biogenesis ...Ribosome biogenesis GTPase RsgA / EngC GTPase domain profile. / RsgA GTPase domain / RsgA GTPase / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain profile. / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / ec:3.6.1.-: / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / ribosomal small subunit biogenesis / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / transcription antitermination / four-way junction DNA binding / negative regulation of translational initiation / positive regulation of translational fidelity / Ribosomal protein S2 signature 2. / endodeoxyribonuclease activity / small ribosomal subunit rRNA binding / DNA-templated transcription, termination / maintenance of translational fidelity / translation repressor activity, mRNA regulatory element binding / ribosomal small subunit assembly / mRNA polyadenylation / mRNA 5'-UTR binding / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S3, bacterial / Ribosomal protein S13, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S19, bacterial-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S5, bacterial-type / Ribosomal protein S16 domain superfamily / Ribosomal protein S18, conserved site / Ribosomal protein S16 / Ribosomal protein S18 signature. / Ribosomal protein S4, bacterial-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S15, bacterial-type / Ribosomal protein S18 / Ribosomal protein S18 superfamily / Ribosomal protein S6 superfamily / Ribosomal protein S6 / cytoplasmic translation / small ribosomal subunit / Ribosomal protein S12, bacterial-type / Translation elongation factor EF1B/ribosomal protein S6 / K Homology domain / GDP binding / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / Ribosomal protein S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S2 signature 1. / Ribosomal protein S14 signature. / Ribosomal protein S14, conserved site / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S2, conserved site / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain superfamily / Type-2 KH domain profile. / Ribosomal protein S5, C-terminal / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5 / Ribosomal protein S5, N-terminal / Ribosomal protein S19, superfamily / Ribosomal protein S19 signature. / Ribosomal protein S19 conserved site / Ribosomal protein S19/S15 / K Homology domain, type 2 / Ribosomal protein S14 / Ribosomal protein S10 / Ribosomal protein S17, conserved site / 30s ribosomal protein S13, C-terminal / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / Ribosomal protein S17 signature. / Ribosomal protein S13 family profile. / Ribosomal protein S13 / Ribosomal protein S11 signature. / Ribosomal S11, conserved site / Ribosomal protein S9 signature. / Ribosomal protein S7, conserved site / Ribosomal protein S9, conserved site / Ribosomal protein S7 signature. / Ribosomal protein S8 signature. / Ribosomal protein S11 / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S15 signature. / Ribosomal protein S10 domain superfamily
Function and homology information
Specimen sourceEscherichia coli / bacteria / /
MethodElectron microscopy (5.8 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsRazi, A. / Guarne, A. / Ortega, J.
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2017
Title: The cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly.
Authors: Aida Razi / Alba Guarné / Joaquin Ortega
Abstract: Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with ...Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 24, 2017 / Release: Apr 19, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 19, 2017Structure modelrepositoryInitial release
1.1Apr 26, 2017Structure modelDatabase references
1.2May 10, 2017Structure modelDatabase references
1.3Sep 27, 2017Structure modelAuthor supporting evidence / Data collectionem_software / pdbx_audit_support_em_software.name / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: 16S RIBOSOMAL RNA
C: 30S ribosomal protein S3
D: 30S ribosomal protein S4
E: 30S ribosomal protein S5
F: 30S ribosomal protein S6
G: 30S ribosomal protein S7
H: 30S ribosomal protein S8
I: 30S ribosomal protein S9
J: 30S ribosomal protein S10
K: 30S ribosomal protein S11
L: 30S ribosomal protein S12
M: 30S ribosomal protein S13
N: 30S ribosomal protein S14
O: 30S ribosomal protein S15
P: 30S ribosomal protein S16
Q: 30S ribosomal protein S17
R: 30S ribosomal protein S18
S: 30S ribosomal protein S19
T: 30S ribosomal protein S20
B: 30S ribosomal protein S2
Z: Small ribosomal subunit biogenesis GTPase RsgA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)813,84423
Polyers813,12321
Non-polymers7212
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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30S ribosomal protein ... , 19 types, 19 molecules CDEFGHIJKLMNOPQRSTB

#2: Protein/peptide 30S ribosomal protein S3


Mass: 26031.316 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCS9, UniProt:P0A7V3*PLUS
#3: Protein/peptide 30S ribosomal protein S4


Mass: 23514.199 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCR2, UniProt:P0A7V8*PLUS
#4: Protein/peptide 30S ribosomal protein S5


Mass: 17629.398 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:P0A7W3, UniProt:P0A7W1*PLUS
#5: Protein/peptide 30S ribosomal protein S6 / Small ribosomal subunit protein bS6


Mass: 15211.058 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:P02358
#6: Protein/peptide 30S ribosomal protein S7 / Small ribosomal subunit protein uS7


Mass: 20055.156 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:P02359
#7: Protein/peptide 30S ribosomal protein S8


Mass: 14146.557 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCS1
#8: Protein/peptide 30S ribosomal protein S9


Mass: 14886.270 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MBZ1
#9: Protein/peptide 30S ribosomal protein S10


Mass: 11755.597 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCT6, UniProt:P0A7R5*PLUS
#10: Protein/peptide 30S ribosomal protein S11


Mass: 13870.975 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCR3
#11: Protein/peptide 30S ribosomal protein S12


Mass: 13768.157 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCV7
#12: Protein/peptide 30S ribosomal protein S13


Mass: 13128.467 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:P0A7T1, UniProt:P0A7S9*PLUS
#13: Protein/peptide 30S ribosomal protein S14


Mass: 11606.560 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCS2, UniProt:P0AG59*PLUS
#14: Protein/peptide 30S ribosomal protein S15


Mass: 10319.882 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:Q8X9M2, UniProt:P0ADZ4*PLUS
#15: Protein/peptide 30S ribosomal protein S16


Mass: 9207.572 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MIU7, UniProt:P0A7T3*PLUS
#16: Protein/peptide 30S ribosomal protein S17


Mass: 9724.491 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCS6
#17: Protein/peptide 30S ribosomal protein S18


Mass: 9005.472 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MLK7
#18: Protein/peptide 30S ribosomal protein S19


Mass: 10455.355 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MCT1, UniProt:P0A7U3*PLUS
#19: Protein/peptide 30S ribosomal protein S20


Mass: 9708.464 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MAE3, UniProt:P0A7U7*PLUS
#20: Protein/peptide 30S ribosomal protein S2


Mass: 26781.670 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: UniProt:B7MBF0, UniProt:P0A7V0*PLUS

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RNA chain / Protein/peptide , 2 types, 2 molecules AZ

#1: RNA chain 16S RIBOSOMAL RNA


Mass: 494971.188 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: GenBank:1095872043
#21: Protein/peptide Small ribosomal subunit biogenesis GTPase RsgA


Mass: 37345.172 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Gene: rsgA, engC, yjeQ, b4161, JW4122 / Production host: Escherichia coli / References: UniProt:P39286, EC:3.6.1.-

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Non-polymers , 2 types, 2 molecules

#22: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Formula: Zn / : Zinc
#23: Chemical ChemComp-GGM / 3'-O-(N-methylanthraniloyl)-beta:gamma-imidoguanosine-5'-triphosphate / MANT-GMPPNP


Mass: 655.343 Da / Num. of mol.: 1 / Formula: C18H24N7O14P3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Structure of the 30S subunit in complex with YjeQ GTPase
Type: COMPLEX
Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21
Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: C-flat CFT-222C
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 25000 / Calibrated magnification: 34482 / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2 mm / C2 aperture diameter: 50 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 0.5 sec. / Electron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 30 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategory
9RELION1.4INITIAL EULER ASSIGNMENT
10RELION1.4FINAL EULER ASSIGNMENT
11RELION1.4CLASSIFICATION
13CootMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 487018
3D reconstructionResolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 130462 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER

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