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- PDB-5lmv: Structure of bacterial 30S-IF1-IF2-IF3-mRNA-tRNA translation pre-... -

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Basic information

Entry
Database: PDB / ID: 5lmv
TitleStructure of bacterial 30S-IF1-IF2-IF3-mRNA-tRNA translation pre-initiation complex(state-III)
Components
  • (30S ribosomal protein ...) x 20
  • (Translation initiation factor IF- ...) x 3
  • 16S ribosomal RNA
  • mRNAMessenger RNA
  • tRNAi
KeywordsRIBOSOME / ribosome / translation / initiation factors / 30S / IF1 / IF2 / IF3 / tRNAi / PIC / Thermus thermophilus
Function / homology30S ribosomal protein Thx / Translation initiation factor IF-2, domain 3 superfamily / Ribosomal protein S20 superfamily / Translation initiation factor 3 (IF-3), N-terminal domain superfamily / Translation initiation factor 3 (IF-3), C-terminal domain superfamily / Ribosomal protein S7 domain superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein S18 superfamily / Ribosomal protein S11 superfamily / Ribosomal protein S8 superfamily ...30S ribosomal protein Thx / Translation initiation factor IF-2, domain 3 superfamily / Ribosomal protein S20 superfamily / Translation initiation factor 3 (IF-3), N-terminal domain superfamily / Translation initiation factor 3 (IF-3), C-terminal domain superfamily / Ribosomal protein S7 domain superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein S18 superfamily / Ribosomal protein S11 superfamily / Ribosomal protein S8 superfamily / RNA-binding S4 domain superfamily / Elongation factor Tu GTP binding domain / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S12/S23 / Ribosomal protein S7p/S5e / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S6 superfamily / Ribosomal protein S14p/S29e / Translation initiation factor IF- 2, domain 3 / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S6, conserved site / Ribosomal protein S19 conserved site / Ribosomal protein S4/S9 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S14, type Z / Ribosomal protein S19, superfamily / Ribosomal protein S6 signature. / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S7 domain / Ribosomal protein S16 domain superfamily / P-loop containing nucleoside triphosphate hydrolase / 30s ribosomal protein S13, C-terminal / Ribosomal protein S10 domain / 30S ribosomal protein / Ribosomal protein S19 / Ribosomal protein S15 / Ribosomal protein S9, conserved site / Ribosomal protein S18 signature. / 30S ribosomal protein Thx / Ribosomal protein S7 signature. / Ribosomal protein S8 signature. / Ribosomal protein S11 signature. / Ribosomal protein S12 signature. / Ribosomal protein S17 signature. / Ribosomal protein S19 signature. / KH domain / Ribosomal protein S9 signature. / Ribosomal protein S10 signature. / Ribosomal protein S15 signature. / Ribosomal protein S14 signature. / Ribosomal protein S3 signature. / Ribosomal protein S5 signature. / Ribosomal protein S4 signature. / Translation-initiation factor 2 / Translation initiation factor IF-3, N-terminal domain / Ribosomal protein S2 / Ribosomal protein S13/S18 / Ribosomal protein S5, N-terminal domain / Ribosomal protein S10p/S20e / Ribosomal protein S17 / Ribosomal protein S9/S16 / Ribosomal protein S8 / Ribosomal protein S11 / Translation initiation factor IF-3, C-terminal domain / Translation initiation factor IF-2, N-terminal region / Ribosomal protein S16 / Ribosomal protein S18 / Translation initiation factor 1A / IF-1 / Ribosomal protein S6 / S4 domain / Ribosomal protein S20 / Ribosomal protein S5, C-terminal domain / Ribosomal protein S7, conserved site / Ribosomal protein S5 domain 2-type fold / Ribosomal protein S2 signature 1. / Ribosomal protein S4/S9, N-terminal / Translation initiation factor 3 / Ribosomal protein S3, C-terminal / Ribosomal protein S18 / Ribosomal protein S10 / Ribosomal protein S2 / Ribosomal protein S13 / Ribosomal protein S11 / Ribosomal protein S5 / Ribosomal protein S19/S15 / Ribosomal protein S20 / RNA-binding S4 domain / K Homology domain, type 2 / K Homology domain / Translation initiation factor IF-1 / Ribosomal protein S14 / Transcription factor, GTP-binding domain / 30S ribosomal protein S17 / Initiation factor 2 signature. / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / S4 RNA-binding domain profile. / S5 double stranded RNA-binding domain profile. / S1 domain IF1 type profile. / Type-2 KH domain profile.
Function and homology information
Specimen sourceThermus thermophilus HB8 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.9 Å resolution
AuthorsHussain, T. / Llacer, J.L. / Wimberly, B.T. / Ramakrishnan, V.
CitationJournal: Cell / Year: 2016
Title: Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.
Authors: Tanweer Hussain / Jose L Llácer / Brian T Wimberly / Jeffrey S Kieft / V Ramakrishnan
Abstract: In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 ...In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 1, 2016 / Release: Oct 5, 2016
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 5, 2016Structure modelrepositoryInitial release
1.1Aug 2, 2017Structure modelData collection / Derived calculationsem_software / pdbx_struct_conn_angle / struct_conn_em_software.name / _em_software.version

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Structure visualization

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Assembly

Deposited unit
A: 16S ribosomal RNA
B: 30S ribosomal protein S2
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 type Z
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
V: 30S ribosomal protein Thx
W: Translation initiation factor IF-1
X: Translation initiation factor IF-3
Y: mRNA
Z: tRNAi
a: Translation initiation factor IF-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)913,49731
Polyers913,14026
Non-polymers3575
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)118270
ΔGint (kcal/M)-953
Surface area (Å2)308010
MethodPISA

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Components

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RNA chain , 3 types, 3 molecules AYZ

#1: RNA chain 16S ribosomal RNA /


Mass: 493958.281 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: GenBank: 55771382
#24: RNA chain mRNA / Messenger RNA


Mass: 13466.019 Da / Num. of mol.: 1 / Source: (synth.) Thermus thermophilus HB8 (bacteria)
#25: RNA chain tRNAi


Mass: 24861.938 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli (E. coli)

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

#2: Protein/peptide 30S ribosomal protein S2 /


Mass: 29317.703 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80371
#3: Protein/peptide 30S ribosomal protein S3 /


Mass: 26751.076 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80372
#4: Protein/peptide 30S ribosomal protein S4 /


Mass: 24373.447 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80373
#5: Protein/peptide 30S ribosomal protein S5 /


Mass: 17583.416 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHQ5
#6: Protein/peptide 30S ribosomal protein S6 / / TS9


Mass: 11988.753 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SLP8
#7: Protein/peptide 30S ribosomal protein S7 /


Mass: 18050.973 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P17291
#8: Protein/peptide 30S ribosomal protein S8 /


Mass: 15868.570 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHQ2, UniProt: P0DOY9*PLUS
#9: Protein/peptide 30S ribosomal protein S9 /


Mass: 14410.614 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80374
#10: Protein/peptide 30S ribosomal protein S10 /


Mass: 11954.968 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHN7
#11: Protein/peptide 30S ribosomal protein S11 /


Mass: 13737.868 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80376
#12: Protein/peptide 30S ribosomal protein S12 /


Mass: 14637.384 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHN3
#13: Protein/peptide 30S ribosomal protein S13 /


Mass: 14338.861 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80377
#14: Protein/peptide 30S ribosomal protein S14 type Z / Ribosome


Mass: 7158.725 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHQ1, UniProt: A0A0N0BLP2*PLUS
#15: Protein/peptide 30S ribosomal protein S15 /


Mass: 10578.407 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SJ76
#16: Protein/peptide 30S ribosomal protein S16 /


Mass: 10409.983 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SJH3
#17: Protein/peptide 30S ribosomal protein S17 /


Mass: 12325.655 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHP7, UniProt: A0A0N0BLS5*PLUS
#18: Protein/peptide 30S ribosomal protein S18 /


Mass: 10258.299 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SLQ0
#19: Protein/peptide 30S ribosomal protein S19 /


Mass: 10605.464 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHP2
#20: Protein/peptide 30S ribosomal protein S20 /


Mass: 11736.143 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80380
#21: Protein/peptide 30S ribosomal protein Thx / Ribosome / S31


Mass: 3350.030 Da / Num. of mol.: 1 / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SIH3

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Translation initiation factor IF- ... , 3 types, 3 molecules WXa

#22: Protein/peptide Translation initiation factor IF-1


Mass: 8247.664 Da / Num. of mol.: 1 / Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: infA, TTHA1669 / Plasmid name: pET13a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SHR1
#23: Protein/peptide Translation initiation factor IF-3


Mass: 19904.377 Da / Num. of mol.: 1 / Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: infC, TTHA0551 / Plasmid name: pET13a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SKU2
#26: Protein/peptide Translation initiation factor IF-2


Mass: 63265.367 Da / Num. of mol.: 1 / Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: infB, TTHA0699 / Plasmid name: pET30a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P48515

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Non-polymers , 3 types, 5 molecules

#27: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc
#28: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Formula: Mg / Magnesium
#29: Chemical ChemComp-FME / N-FORMYLMETHIONINE


Mass: 177.221 Da / Num. of mol.: 1 / Formula: C6H11NO3S / N-Formylmethionine

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: 30S-IF1-IF2-IF3-mRNA-tRNA pre-initiation complex (state-III)
Type: RIBOSOME
Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26
Source: MULTIPLE SOURCES
Molecular weightValue: 0.9129 MDa / Experimental value: NO
Buffer solutionpH: 6.5
Buffer component
IDConc.NameBuffer ID
110 mMMES1
25 mMMagnessium acetate1
350 mMPotassium chloride1
410 mMAmmonium chloride1
56 mMBeta mercaptoethanol1
SpecimenConc.: 0.065 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 / Calibrated magnification: 104478 / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 kelvins / Temperature (min): 90 kelvins
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 30 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Number of grids imaged: 1 / Number of real images: 3200
Image scansMovie frames/image: 25

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Processing

EM software
IDNameVersionCategory
2EMANparticle selection
3EPUimage acquisition
5CTFFIND3CTF correction
8UCSF Chimera1.1model fitting
9Coot0.8model fitting
11RELION1.4initial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
15REFMAC5.8model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 803433
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 26324 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: RECIPROCAL / Target criteria: FSC

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