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- PDB-4x4u: Crystal structure of the A.fulgidus CCA-adding enzyme in complex ... -

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Basic information

Entry
Database: PDB / ID: 4x4u
TitleCrystal structure of the A.fulgidus CCA-adding enzyme in complex with a human MenBeta minihelix ending in CCACC
Components
  • CCA-adding enzyme
  • RNA (5'-D(*GP*G)-3')
  • human MenBeta minihelix
KeywordsRNA BINDING PROTEIN / protein-RNA complex / tRNA / non-coding RNA / CCA-adding enzyme / nucleotidyltransferase / ncRNA
Function / homology
Function and homology information


CCA tRNA nucleotidyltransferase / tRNA surveillance / CCACCA tRNA nucleotidyltransferase activity / CCA tRNA nucleotidyltransferase activity / 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 / Polymerase, nucleotidyl transferase domain / Poly(a)-polymerase, middle 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
DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / DI(HYDROXYETHYL)ETHER / 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: 2.7 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: Derived calculations
Revision 1.3Nov 22, 2017Group: 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_oper_list / 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_oper_list.symmetry_operation
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
B: human MenBeta minihelix
C: CCA-adding enzyme
D: human MenBeta minihelix
E: CCA-adding enzyme
F: CCA-adding enzyme
G: human MenBeta minihelix
H: RNA (5'-D(*GP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)252,67717
Polymers250,9948
Non-polymers1,6839
Water1,78399
1
A: CCA-adding enzyme
B: human MenBeta minihelix
H: RNA (5'-D(*GP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,0788
Polymers66,2043
Non-polymers8745
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: CCA-adding enzyme
D: human MenBeta minihelix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,6653
Polymers65,5592
Non-polymers1061
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
E: CCA-adding enzyme
F: CCA-adding enzyme
G: human MenBeta minihelix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,9346
Polymers119,2313
Non-polymers7033
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)114.988, 84.049, 135.524
Angle α, β, γ (deg.)90.000, 103.420, 90.000
Int Tables number4
Space group name H-MP1211

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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 (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 BDGH

#2: RNA chain human MenBeta minihelix


Mass: 11886.064 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: RNA chain RNA (5'-D(*GP*G)-3')


Mass: 645.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 4 types, 108 molecules

#4: Chemical ChemComp-APC / DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / ALPHA,BETA-METHYLENEADENOSINE-5'-TRIPHOSPHATE


Mass: 505.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H18N5O12P3 / Comment: AMP-CPP, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 99 / Source method: isolated from a natural source / Formula: H2O

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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: 2.54 Å3/Da / Density % sol: 51.54 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / Details: 18.9% PEG3350, 0.27M NaK tartrate

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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: May 3, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 2.7→30 Å / Num. obs: 68272 / % possible obs: 97.6 % / Redundancy: 2.8 % / Biso Wilson estimate: 65.2 Å2 / Rmerge(I) obs: 0.075 / Χ2: 1.091 / Net I/av σ(I): 15.12 / Net I/σ(I): 8.9 / Num. measured all: 191792
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2% possible all
2.7-2.752.50.36731890.58692.8
2.75-2.82.80.34334180.698.4
2.8-2.852.90.31234180.60698.4
2.85-2.912.90.27234520.65198.1
2.91-2.972.80.2233690.70897.9
2.97-3.042.70.1834100.75898.3
3.04-3.122.90.15934530.81398.6
3.12-3.22.80.13334170.84898.5
3.2-3.292.80.11834040.99498.3
3.29-3.42.80.10634571.18298
3.4-3.522.90.09633851.31797.8
3.52-3.662.90.08734151.3997.7
3.66-3.832.70.08234071.45997.7
3.83-4.032.90.07634261.51898.1
4.03-4.282.80.07134151.41898
4.28-4.612.90.0734411.44397.5
4.61-5.072.80.06734101.4797.4
5.07-5.82.90.06534581.35598.3
5.8-7.32.80.06234491.27697
7.3-302.80.05234791.26895.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data reduction
PDB_EXTRACT3.14data extraction
PHASERphasing
PHENIX(phenix.refine: 1.8.2_1309)refinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.7→29.793 Å / FOM work R set: 0.8293 / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.4 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2334 3410 5.02 %
Rwork0.1827 64464 -
obs0.1853 67874 97.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 534.48 Å2 / Biso mean: 84.59 Å2 / Biso min: 29.1 Å2
Refinement stepCycle: final / Resolution: 2.7→29.793 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14497 1644 262 99 16502
Biso mean--118.12 57.19 -
Num. residues----1820
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00516766
X-RAY DIFFRACTIONf_angle_d0.80522914
X-RAY DIFFRACTIONf_chiral_restr0.0442460
X-RAY DIFFRACTIONf_plane_restr0.0042672
X-RAY DIFFRACTIONf_dihedral_angle_d12.2886677
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 24

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7-2.73860.29881410.24082671281298
2.7386-2.77940.29831430.22992672281598
2.7794-2.82280.29111370.23852684282198
2.8228-2.8690.31721450.23872696284199
2.869-2.91850.29991390.23532654279398
2.9185-2.97150.28691380.23182670280898
2.9715-3.02860.2591420.23282704284698
3.0286-3.09040.31241490.22212671282098
3.0904-3.15750.28621340.21432689282399
3.1575-3.23080.27391490.20862720286998
3.2308-3.31150.23641350.2092659279498
3.3115-3.4010.25831440.20422713285798
3.401-3.50090.28141440.20442642278698
3.5009-3.61370.2561370.19122670280798
3.6137-3.74260.23281430.18582678282198
3.7426-3.89220.20471420.1672684282698
3.8922-4.06890.21791450.17012702284798
4.0689-4.28280.22141410.15952677281898
4.2828-4.55030.20581400.15182696283698
4.5503-4.90020.20621480.14012674282297
4.9002-5.39070.19851450.15412708285398
5.3907-6.16470.22211440.17662721286598
6.1647-7.74420.21221410.1862687282897
7.7442-29.79490.21791440.18032722286695
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.53340.25620.92360.61160.1030.70910.26820.6226-0.2328-0.195-0.0052-0.17470.19350.197700.81020.26460.05230.8808-0.03140.6717182.36955.3394129.1417
21.7912-0.28270.10010.26260.02342.58550.29080.41750.1218-0.1977-0.0493-0.06510.1344-0.04020.02180.38310.10910.05890.49210.15790.3765179.378121.4726145.5107
30.6998-0.4809-0.29150.2616-0.14661.70820.06580.0761-0.0263-0.0576-0.01160.05320.55680.22390.00420.48350.0671-0.11660.1887-0.07750.4571185.22792.7895174.773
40.29230.0081-0.88590.4749-1.30872.69020.4590.31360.3620.67140.68930.22430.7292-1.5921.8491.6039-0.17260.03941.27230.00431.0095167.5396-5.4305168.8814
50.00760.02490.0278-0.01490.04760.00480.4210.44080.0771-1.38380.4591-0.42680.70320.95160.00061.6618-0.2144-0.12651.82670.02471.3935173.53862.4913144.6501
6-0.1214-0.6324-0.22850.68470.49410.4366-0.26780.36080.02-0.27660.2009-0.2668-1.06920.5919-0.00911.067-0.5460.09861.2705-0.05240.7266250.1078-0.4431130.8981
72.1704-0.08010.43772.96330.56842.5769-0.37450.1901-0.3283-0.47040.35770.0532-0.53780.2153-0.20320.6621-0.233-0.02460.536-0.10190.093230.6451-3.1511141.4267
81.25110.66670.18680.50110.35361.44-0.21010.0794-0.0716-0.13590.1952-0.067-0.49630.39140.02630.3748-0.07470.00390.4043-0.04780.261239.765813.1609173.0774
90.0665-0.0320.08040.0387-0.01970.17210.69150.12110.4308-0.02441.254-1.07910.78661.53480.00293.0005-0.2490.66741.59380.06791.6509249.5016-3.8961158.8063
10-0.18020.0492-0.11580.1476-0.16980.12440.1774-0.98790.5340.43710.90540.06440.1586-0.1357-0.00042.1840.13930.42761.9960.09441.0449251.3746-4.8591181.7042
112.4367-0.3106-1.0940.04110.28211.2644-0.7542-1.6755-1.1187-1.46851.05760.43191.82390.2217-0.02771.95360.02030.27781.27850.11861.2222249.9296-5.0187164.4793
120.09050.8114-1.15921.5594-1.57631.0352-0.25680.3354-0.0215-0.14760.22160.1723-0.01410.0767-0.03140.3605-0.00490.01830.5336-0.09720.5695213.672-21.3489167.5288
131.45251.2517-1.03140.7512-0.65531.4853-0.0535-0.0257-0.11690.0826-0.0341-0.08760.02010.2313-0.00010.28710.08730.00450.3313-0.05550.4647214.5231-34.0163184.9315
140.2135-0.01120.2534-0.1226-0.0690.75310.1082-0.3351-0.3082-0.130.168-0.27930.4889-0.34480.01310.58960.0198-0.06580.3323-0.00750.6406199.6828-42.465184.4011
150.7194-0.2125-0.43440.29540.20761.6520.1264-0.00290.0936-0.1113-0.01730.033-0.0181-0.16110.00020.3881-0.014-0.0320.17230.00980.453184.0828-21.0462203.1731
162.311.2972-0.81790.866-0.6371.05490.0105-0.0270.35330.0513-0.07480.37-0.09350.2727-0.01720.3631-0.02390.04170.437-0.05830.5287208.4207-6.1677228.1615
171.41060.6843-0.77531.4911-0.93531.30260.0562-0.0425-0.02470.0135-0.15880.16470.16630.1479-0.00040.27020.02760.04420.52070.03780.2731225.2892-11.3775216.8708
180.321-0.7097-0.31842.0080.40680.34110.3879-0.00210.5128-0.1535-0.46030.1063-0.48660.3022-0.04410.3837-0.10530.0290.69910.02670.5084233.9285.3488213.1576
190.4231-0.1994-0.56340.4165-0.10172.31290.008-0.0316-0.0578-0.05070.04510.1905-0.3025-0.3076-0.00350.28040.0772-0.0180.4483-0.03430.448217.809212.1405185.9361
200.3710.18060.50870.31360.10260.20830.4217-0.4120.8334-0.14370.56540.59160.41950.05290.03350.9766-0.05180.10131.00820.02560.8279206.6789-10.8493199.9622
210.03440.07290.21130.15360.16860.06210.0428-0.00850.43390.06081.16810.790.34360.5637-0.00051.0081-0.00660.07481.0785-0.00770.9857209.6654-10.0198195.4255
220.0043-0.0123-0.00830.0133-0.01620.0089-0.15640.02370.0159-0.34220.38640.53590.134-0.184-0.00041.92450.6164-0.00511.75550.21581.3101160.90970.8464183.643
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 144 )A0
2X-RAY DIFFRACTION2chain 'A' and (resid 145 through 263 )A0
3X-RAY DIFFRACTION3chain 'A' and (resid 264 through 437 )A0
4X-RAY DIFFRACTION4chain 'B' and (resid 1 through 24 )B0
5X-RAY DIFFRACTION5chain 'B' and (resid 31 through 37 )B0
6X-RAY DIFFRACTION6chain 'C' and (resid 1 through 129 )C0
7X-RAY DIFFRACTION7chain 'C' and (resid 130 through 263 )C0
8X-RAY DIFFRACTION8chain 'C' and (resid 264 through 437 )C0
9X-RAY DIFFRACTION9chain 'D' and (resid 1 through 6 )D0
10X-RAY DIFFRACTION10chain 'D' and (resid 7 through 16 )D0
11X-RAY DIFFRACTION11chain 'D' and (resid 20 through 34 )D0
12X-RAY DIFFRACTION12chain 'E' and (resid 1 through 144 )E0
13X-RAY DIFFRACTION13chain 'E' and (resid 145 through 231 )E0
14X-RAY DIFFRACTION14chain 'E' and (resid 232 through 263 )E0
15X-RAY DIFFRACTION15chain 'E' and (resid 264 through 437 )E0
16X-RAY DIFFRACTION16chain 'F' and (resid 2 through 126 )F0
17X-RAY DIFFRACTION17chain 'F' and (resid 127 through 231 )F0
18X-RAY DIFFRACTION18chain 'F' and (resid 232 through 263 )F0
19X-RAY DIFFRACTION19chain 'F' and (resid 264 through 437 )F0
20X-RAY DIFFRACTION20chain 'G' and (resid 1 through 23 )G0
21X-RAY DIFFRACTION21chain 'G' and (resid 24 through 31 )G0
22X-RAY DIFFRACTION22chain 'H' and (resid 1 through 2 )H0

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