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- PDB-4x4n: Crystal structure of the A.fulgidus CCA-adding enzyme in complex ... -

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Basic information

Entry
Database: PDB / ID: 4x4n
TitleCrystal structure of the A.fulgidus CCA-adding enzyme in complex with a G70A arginyl-tRNA minihelix
Components
  • CCA-adding enzyme
  • G70A tRNA minihelix
  • RNA (5'-D(P*CP*G)-3')
Keywordstransferase/RNA / protein-RNA complex / tRNA / non-coding RNA / CCA-adding enzyme / nucleotidyltransferase / ncRNA / transferase-RNA complex
Function / homology
Function and homology information


tRNA surveillance / CCACCA tRNA nucleotidyltransferase activity / CCA tRNA nucleotidyltransferase / tRNA 3'-terminal CCA addition / RNA repair / tRNA binding / magnesium ion binding / ATP binding
Similarity search - Function
CCA tRNA nucleotidyltransferase, domain 2 / Archaeal tRNA CCA-adding enzyme catalytic domain / tRNA nucleotidyltransferase, archaea / tRNA nucleotidyltransferase, substrate binding / tRNA nucleotidyltransferase, domain 2 / : / tRNA nucleotidyltransferase, second domain / CCA-adding enzyme, C-terminal domain / Poly(A) polymerase predicted RNA binding domain / Nucleotidyltransferase, class I-like, C-terminal ...CCA tRNA nucleotidyltransferase, domain 2 / Archaeal tRNA CCA-adding enzyme catalytic domain / tRNA nucleotidyltransferase, archaea / tRNA nucleotidyltransferase, substrate binding / tRNA nucleotidyltransferase, domain 2 / : / tRNA nucleotidyltransferase, second domain / CCA-adding enzyme, C-terminal domain / Poly(A) polymerase predicted RNA binding domain / Nucleotidyltransferase, class I-like, C-terminal / 2-5OAS/ClassI-CCAase, nucleotidyltransferase domain / Poly(a)-polymerase, middle domain / Polymerase, nucleotidyl transferase domain / Nucleotidyltransferase domain / Beta Polymerase, domain 2 / Beta Polymerase; domain 2 / Nucleotidyltransferase superfamily / Alpha-Beta Plaits / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-MONOPHOSPHATE / GUANOSINE / RNA / RNA (> 10) / CCA-adding enzyme
Similarity search - Component
Biological speciesArchaeoglobus fulgidus (archaea)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.953 Å
AuthorsKuhn, C.-D. / Joshua-Tor, L.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2015
Title: On-Enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-Adding Enzyme.
Authors: Kuhn, C.D. / Wilusz, J.E. / Zheng, Y. / Beal, P.A. / Joshua-Tor, L.
History
DepositionDec 3, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 11, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 18, 2015Group: Database references
Revision 1.2Feb 25, 2015Group: Database references
Revision 1.3Nov 22, 2017Group: Advisory / Derived calculations ...Advisory / Derived calculations / Refinement description / Source and taxonomy
Category: entity_src_gen / pdbx_entity_src_syn ...entity_src_gen / pdbx_entity_src_syn / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_oper_list / pdbx_validate_close_contact / software
Item: _entity_src_gen.pdbx_alt_source_flag / _pdbx_entity_src_syn.pdbx_alt_source_flag ..._entity_src_gen.pdbx_alt_source_flag / _pdbx_entity_src_syn.pdbx_alt_source_flag / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_assembly_prop.type / _pdbx_struct_assembly_prop.value / _pdbx_struct_oper_list.symmetry_operation / _software.name
Revision 1.4Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Dec 27, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CCA-adding enzyme
G: G70A tRNA minihelix
B: G70A tRNA minihelix
C: CCA-adding enzyme
D: G70A tRNA minihelix
E: CCA-adding enzyme
F: CCA-adding enzyme
I: RNA (5'-D(P*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)247,32515
Polymers246,2028
Non-polymers1,1237
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18890 Å2
ΔGint-135 kcal/mol
Surface area95020 Å2
MethodPISA
Unit cell
Length a, b, c (Å)115.416, 84.688, 135.565
Angle α, β, γ (deg.)90.000, 103.140, 90.000
Int Tables number4
Space group name H-MP1211
Detailsbiological units are chains A+B and C+D

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Components

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Protein , 1 types, 4 molecules ACEF

#1: Protein
CCA-adding enzyme / CCA tRNA nucleotidyltransferase / tRNA CCA-pyrophosphorylase / tRNA adenylyl-/cytidylyl- ...CCA tRNA nucleotidyltransferase / tRNA CCA-pyrophosphorylase / tRNA adenylyl-/cytidylyl- transferase / tRNA nucleotidyltransferase / tRNA-NT


Mass: 53672.484 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126) (archaea)
Strain: ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126
Gene: cca, AF_2156 / Plasmid: pET22 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)RIPL
References: UniProt: O28126, CCA tRNA nucleotidyltransferase

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RNA chain , 2 types, 4 molecules GBDI

#2: RNA chain G70A tRNA minihelix


Mass: 10302.164 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: RNA chain RNA (5'-D(P*CP*G)-3')


Mass: 605.430 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 4 types, 7 molecules

#4: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-GMP / GUANOSINE


Mass: 283.241 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H13N5O5
#6: Chemical ChemComp-5GP / GUANOSINE-5'-MONOPHOSPHATE


Mass: 363.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O8P
#7: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.62 Å3/Da / Density % sol: 53.06 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 7.75
Details: 18.6% PEG4000, 0.2M tri-Lithium Citrate, 0.05M Hepes pH 7.75, 0.08M Ammonium Sulfate
PH range: 7.75

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 1.1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 25, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 2.95→30 Å / Num. obs: 53329 / % possible obs: 99.8 % / Redundancy: 3.3 % / Biso Wilson estimate: 67.8 Å2 / Rmerge(I) obs: 0.056 / Χ2: 0.771 / Net I/av σ(I): 18.586 / Net I/σ(I): 8.1 / Num. measured all: 173434
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2% possible all
2.95-33.30.34126260.63699.9
3-3.063.10.29626390.67199.6
3.06-3.113.30.24326470.65599.8
3.11-3.183.30.21926300.66999.9
3.18-3.253.30.18126890.691100
3.25-3.323.10.14626190.73399.9
3.32-3.43.40.12726780.741100
3.4-3.53.30.11326450.81599.8
3.5-3.63.30.09326310.77799.7
3.6-3.723.10.08426730.91699.9
3.72-3.853.30.07126690.90999.9
3.85-43.30.06526520.96699.7
4-4.183.20.05526650.97100
4.18-4.43.40.0526600.958100
4.4-4.683.30.04526800.8799.9
4.68-5.043.20.04226780.81599.9
5.04-5.543.20.0426700.70899.9
5.54-6.343.40.0426980.65199.9
6.34-7.963.20.03327040.61199.4
7.96-303.10.02527760.661100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE: 1.8.2_1309)refinement
HKL-2000data reduction
PDB_EXTRACT3.14data extraction
PHASERphasing
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.953→29.885 Å / FOM work R set: 0.8076 / SU ML: 0.4 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.31 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2489 2669 5.01 %
Rwork0.1914 50637 -
obs0.1943 53306 99.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 231.54 Å2 / Biso mean: 46.99 Å2 / Biso min: 2.15 Å2
Refinement stepCycle: final / Resolution: 2.953→29.885 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14507 1818 168 0 16493
Biso mean--74.48 --
Num. residues----1828
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00217308
X-RAY DIFFRACTIONf_angle_d0.55923764
X-RAY DIFFRACTIONf_chiral_restr0.0342581
X-RAY DIFFRACTIONf_plane_restr0.0032694
X-RAY DIFFRACTIONf_dihedral_angle_d11.4536918
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 19

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.9533-3.0070.33521260.25362310243687
3.007-3.06480.32141340.262726842818100
3.0648-3.12730.3761410.246526472788100
3.1273-3.19520.33661410.2426642805100
3.1952-3.26940.30091380.24226472785100
3.2694-3.35110.28581490.233826772826100
3.3511-3.44160.25141380.215826552793100
3.4416-3.54270.27781390.221126712810100
3.5427-3.65680.29211360.215826852821100
3.6568-3.78730.31431480.208526702818100
3.7873-3.93860.23851370.193726622799100
3.9386-4.11740.24651400.187227172857100
4.1174-4.33390.22051410.173826582799100
4.3339-4.60450.25931440.169926952839100
4.6045-4.95850.22281410.154426962837100
4.9585-5.45480.21131420.164726932835100
5.4548-6.23790.23671430.183127052848100
6.2379-7.83560.21651440.183727142858100
7.8356-29.88630.19761470.168327872934100

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