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- PDB-5u4j: Structural Basis of Co-translational Quality Control by ArfA and ... -

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Entry
Database: PDB / ID: 5u4j
TitleStructural Basis of Co-translational Quality Control by ArfA and RF2 Bound to Ribosome
DescriptorRibosome
KeywordsRIBOSOME / Ribosome / ArfA / RF2 / nonstop translation
Specimen sourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
MethodElectron microscopy (3.7 Å resolution / Particle / Single particle)
AuthorsZeng, F. / Chen, Y. / Remis, J. / Shekhar, M. / Phillips, J.C. / Tajkhorshid, E. / Jin, H.
CitationNature, 2017, 541, 554-557

Nature, 2017, 541, 554-557 Yorodumi Papers
Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome.
Fuxing Zeng / Yanbo Chen / Jonathan Remis / Mrinal Shekhar / James C Phillips / Emad Tajkhorshid / Hong Jin

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 4, 2016 / Release: Jan 11, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 11, 2017Structure modelrepositoryInitial release
1.1Jan 25, 2017Structure modelDatabase references
1.2Feb 8, 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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Assembly

Deposited unit
a: 16S rRNA
A: 23S rRNA
c: 30S ribosomal protein S3
d: 30S ribosomal protein S4
e: 30S ribosomal protein S5
z: mRNA
x: P-site tRNA fMet
l: 30S ribosomal protein S12
v: Peptide chain release factor 2
w: Alternative ribosome-rescue factor A


Theoretical massNumber of molelcules
Total (without water)1,600,33210
Polyers1,600,33210
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 4 types, 4 molecules aAzx

#1: RNA chain16S rRNA


Mass: 497075.812 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: GenBank: 817573384
#2: RNA chain23S rRNA


Mass: 941790.625 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli / References: GenBank: 42756
#6: RNA chainmRNA


Mass: 5922.644 Da / Num. of mol.: 1 / Source: (synth.) Escherichia coli
#7: RNA chainP-site tRNA fMet


Mass: 24786.785 Da / Num. of mol.: 1 / Source: (synth.) Escherichia coli / References: GenBank: 687670942

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30S ribosomal protein ... , 4 types, 4 molecules cdel

#3: Polypeptide(L)30S ribosomal protein S3


Mass: 26031.316 Da / Num. of mol.: 1 / Details: Gene Name(s): rpsC b3314 JW3276 / Source: (natural) Escherichia coli / References: UniProt: P0A7V3

Cellular component

Molecular function

Biological process

#4: Polypeptide(L)30S ribosomal protein S4


Mass: 23514.199 Da / Num. of mol.: 1 / Details: Gene Name(s): rpsD ramA b3296 JW3258 / Source: (natural) Escherichia coli / References: UniProt: P0A7V8

Cellular component

Molecular function

Biological process

#5: Polypeptide(L)30S ribosomal protein S5


Mass: 17629.398 Da / Num. of mol.: 1 / Details: Gene Name(s): rpsE spc b3303 JW3265 / Source: (natural) Escherichia coli / References: UniProt: P0A7W1

Cellular component

Molecular function

Biological process

#8: Polypeptide(L)30S ribosomal protein S12


Mass: 13768.157 Da / Num. of mol.: 1 / Details: Gene Name(s): rpsL strA b3342 JW3304 / Source: (natural) Escherichia coli / References: UniProt: P0A7S3

Cellular component

Molecular function

Biological process

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Polypeptide(L) , 2 types, 2 molecules vw

#9: Polypeptide(L)Peptide chain release factor 2 / RF-2


Mass: 43299.844 Da / Num. of mol.: 1 / Details: Gene Name(s): prfB ECK2886 JW5847 supK / Source: (gene. exp.) Escherichia coli / References: UniProt: P07012

Cellular component

Molecular function

  • translation release factor activity, codon specific (GO: 0016149)

Biological process

#10: Polypeptide(L)Alternative ribosome-rescue factor A


Mass: 6513.396 Da / Num. of mol.: 1 / Details: Gene Name(s): arfA ECK3278 JW3253 yhdL / Source: (gene. exp.) Escherichia coli / References: UniProt: P36675

Molecular function

Biological process

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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

Component
IDNameTypeEntity IDParent IDSourceDetails
1Non-stop ribosomal complex with ArfA and RF2RIBOSOME1, 2, 3, 4, 5, 6, 7, 8, 9, 100MULTIPLE SOURCES
230S subunitCOMPLEX1, 3, 4, 5, 81NATURAL
350S subunitCOMPLEX21NATURAL
4Alternative ribosome-rescue factor AORGANELLE OR CELLULAR COMPONENT101RECOMBINANT
5Peptide chain release factor 2ORGANELLE OR CELLULAR COMPONENT91RECOMBINANT
6P-site tRNA fMetORGANELLE OR CELLULAR COMPONENT71MULTIPLE SOURCESP-site tRNA (fMet) was in vitro transcribed.
7mRNAORGANELLE OR CELLULAR COMPONENT61MULTIPLE SOURCES
823S rRNAORGANELLE OR CELLULAR COMPONENT23NATURAL
916S rRNAORGANELLE OR CELLULAR COMPONENT12NATURAL
1030S ribosomal protein S3ORGANELLE OR CELLULAR COMPONENT32NATURAL
1130S ribosomal protein S4ORGANELLE OR CELLULAR COMPONENT42NATURAL
1230S ribosomal protein S5ORGANELLE OR CELLULAR COMPONENT2NATURAL
1330S ribosomal protein S12ORGANELLE OR CELLULAR COMPONENT2NATURAL
Molecular weight
IDValueUnitsEntity assembly IDExperimental value
11NO
21NO
31NO
46.35KILODALTONS/NANOMETER1NO
540.13KILODALTONS/NANOMETER1NO
61NO
71NO
81NO
91
110
111
112
113
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
22562Escherichia coli
33562Escherichia coli
44562Escherichia coli
55562Escherichia coli
66562Escherichia coli
78562Escherichia coli
89562Escherichia coli
910562Escherichia coli
1011562Escherichia coli
1112562Escherichia coli
1213562Escherichia coli
11562Escherichia coli
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismPlasmidStrain
34562Escherichia colipET28BL21
45562Escherichia colipETNKI-his3C-LIC-KanBL21
56562Escherichia colipUC18
Buffer solutionpH: 7.5
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidC8H18N2O4S1
215mMmagnesium acetateMg(CH3COO)21
3150mMpotassium acetateCH3CO2K1
44mM2-mercapthoethanolHOCH2CH2SH1
52mMspermidineC7H19N31
60.05mMsperminC10H26N41
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-Flat TM Holey Carbon Grid CF-2/0.5-4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins / Details: Grids were blotted for 3.5 s

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Electron microscopy imaging

MicroscopyMicroscope model: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 83822 / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6 sec. / Electron dose: 20 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 30 / Used frames/image: 4-25

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Processing

SoftwareName: REFMAC / Version: 5.8.0155 / Classification: refinement
EM software
IDNameVersionCategoryImage processing IDFitting ID
1RELION1.4PARTICLE SELECTION1
4CTFFIND4CTF CORRECTION1
7ChimeraMODEL FITTING1
9RELION2.0INITIAL EULER ASSIGNMENT1
10RELION2.0FINAL EULER ASSIGNMENT1
12RELION2.0RECONSTRUCTION1
13REFMAC5.8MODEL REFINEMENT1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 155440 / Symmetry type: POINT
Atomic model buildingOverall b value: 108.501 / Ref protocol: FLEXIBLE FIT / Ref space: RECIPROCAL / Target criteria: Average FSC
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLOverall ESU RStereochemistry target valuesSolvent model details
1108.510-0.17-0.46-1.700.01-0.010.160.5960.433550.433553.70458.113976064100.006.6110.0940.086MAXIMUM LIKELIHOOD WITH PHASESPARAMETERS FOR MASK CACLULATION
ELECTRON MICROSCOPY
Number of atoms included #1Total: 11360
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01412278
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg0.9041.51718210
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg3.8525.000517
ELECTRON MICROSCOPYr_dihedral_angle_2_deg41.52622.139187
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.79415.000776
ELECTRON MICROSCOPYr_dihedral_angle_4_deg8.72915.00050
ELECTRON MICROSCOPYr_chiral_restr0.0660.2001954
ELECTRON MICROSCOPYr_gen_planes_refined0.0020.0206637
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.19514.5572089
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it4.14921.8152599
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it1.2869.91610189
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined9.29320376
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 3.7 Å / R factor R work: 1.519 / Lowest resolution: 3.796 Å / Number reflection R free: 0 / Number reflection R work: 295394 / Total number of bins used: 20 / Percent reflection obs: 1

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