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- PDB-4x4v: Crystal structure of the A.fulgidus CCA-adding enzyme in complex ... -

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Basic information

Entry
Database: PDB / ID: 4x4v
TitleCrystal structure of the A.fulgidus CCA-adding enzyme in complex with a human MenBeta minihelix ending in CCACC and AMPcPP
Components
  • CCA-adding enzyme
  • Human MenBeta minihelix ending in CCACC
KeywordsRNA BINDING PROTEIN / protein-RNA complex / tRNA / non-coding RNA / CCA-adding enzyme / nucleotidyltransferase / ncRNA
Function / homology
Function and homology information


CCACCA tRNA nucleotidyltransferase activity / CCA tRNA nucleotidyltransferase / tRNA 3'-terminal CCA addition / tRNA surveillance / 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
GUANOSINE-5'-MONOPHOSPHATE / DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / D(-)-TARTARIC ACID / 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: 2.6 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: Derived calculations / Refinement description / Source and taxonomy
Category: entity_src_gen / pdbx_entity_src_syn ...entity_src_gen / pdbx_entity_src_syn / pdbx_struct_oper_list / software
Item: _entity_src_gen.pdbx_alt_source_flag / _pdbx_entity_src_syn.pdbx_alt_source_flag / _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 / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id

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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 ending in CCACC
C: CCA-adding enzyme
D: Human MenBeta minihelix ending in CCACC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,06116
Polymers131,1174
Non-polymers2,94412
Water3,873215
1
A: CCA-adding enzyme
B: Human MenBeta minihelix ending in CCACC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,46010
Polymers65,5592
Non-polymers1,9018
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5740 Å2
ΔGint-28 kcal/mol
Surface area27090 Å2
MethodPISA
2
C: CCA-adding enzyme
D: Human MenBeta minihelix ending in CCACC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,6016
Polymers65,5592
Non-polymers1,0434
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4260 Å2
ΔGint-36 kcal/mol
Surface area26430 Å2
MethodPISA
3


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15160 Å2
ΔGint-70 kcal/mol
Surface area48360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)111.059, 215.602, 58.629
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212

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Components

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Protein / RNA chain , 2 types, 4 molecules ACBD

#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.: 2
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
#2: RNA chain Human MenBeta minihelix ending in CCACC


Mass: 11886.064 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 6 types, 227 molecules

#3: 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
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-TAR / D(-)-TARTARIC ACID


Mass: 150.087 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H6O6
#6: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#7: Chemical ChemComp-5GP / GUANOSINE-5'-MONOPHOSPHATE


Mass: 363.221 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H14N5O8P
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 215 / 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: 2.68 Å3/Da / Density % sol: 54.05 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / pH: 6.8 / Details: 23.2% PEG3350, 0.2M NaK tartrate, 0.1M MES pH 6.8 / PH range: 6.8

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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: Mar 7, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 2.6→30 Å / Num. obs: 43940 / % possible obs: 98.5 % / Redundancy: 3.3 % / Biso Wilson estimate: 36.59 Å2 / Rmerge(I) obs: 0.093 / Χ2: 1.123 / Net I/av σ(I): 13.125 / Net I/σ(I): 7.1 / Num. measured all: 143132
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2% possible all
2.6-2.642.90.38420040.83790.9
2.64-2.693.20.34621630.85999.8
2.69-2.743.10.32721900.92299.5
2.74-2.83.30.28922160.90999.6
2.8-2.863.40.27721460.91899.5
2.86-2.933.30.24222110.94299.5
2.93-33.30.20421361.0297.6
3-3.083.30.17321671.08198.5
3.08-3.173.30.15821931.12499.8
3.17-3.283.30.13222031.22799.6
3.28-3.393.20.1221881.26299.5
3.39-3.533.40.10722061.22399.2
3.53-3.693.30.09522021.23898.7
3.69-3.883.30.08421751.26198.4
3.88-4.123.30.07622211.33899.5
4.12-4.443.20.06822351.28299.6
4.44-4.893.30.05922411.30498.9
4.89-5.593.30.05622171.29398.2
5.59-7.033.20.05422801.0898.6
7.03-303.10.0423461.23996.2

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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: 2.6→29.68 Å / FOM work R set: 0.8376 / SU ML: 0.3 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 22.98 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2372 2218 5.05 %
Rwork0.1841 41680 -
obs0.1868 43898 98.47 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 177.22 Å2 / Biso mean: 47.8 Å2 / Biso min: 14.61 Å2
Refinement stepCycle: final / Resolution: 2.6→29.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7248 1258 267 215 8988
Biso mean--56.55 33.59 -
Num. residues----933
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0079019
X-RAY DIFFRACTIONf_angle_d0.98912443
X-RAY DIFFRACTIONf_chiral_restr0.0521364
X-RAY DIFFRACTIONf_plane_restr0.0051368
X-RAY DIFFRACTIONf_dihedral_angle_d14.5553626
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 16

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.5941-2.65040.2981340.2222393252791
2.6504-2.7120.3111420.218725552697100
2.712-2.77980.28011300.22226302760100
2.7798-2.85490.29951330.229825892722100
2.8549-2.93890.30721360.2282603273999
2.9389-3.03360.23521320.20362521265397
3.0336-3.14190.22481340.213426062740100
3.1419-3.26760.28561350.203826362771100
3.2676-3.41610.25031270.20562612273999
3.4161-3.59590.27991470.18592609275699
3.5959-3.82080.24671290.17442579270898
3.8208-4.11510.20421340.166526492783100
4.1151-4.52790.21171450.151126532798100
4.5279-5.18010.20281390.13892634277398
5.1801-6.5150.21141680.17692672284099
6.515-29.6820.19841530.18022739289297

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