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- PDB-4x4o: Crystal structure of the A.fulgidus CCA-adding enzyme in complex ... -

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Basic information

Entry
Database: PDB / ID: 4x4o
TitleCrystal structure of the A.fulgidus CCA-adding enzyme in complex with a G70A arginyl-tRNA minihelix and CTP
Components
  • CCA-adding enzyme
  • G70A tRNA 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
CYTIDINE-5'-TRIPHOSPHATE / : / 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: 3.201 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 / diffrn_radiation_wavelength / 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_auth_seq_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: G70A tRNA minihelix
C: CCA-adding enzyme
D: G70A tRNA minihelix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,05915
Polymers129,1704
Non-polymers1,88911
Water362
1
A: CCA-adding enzyme
B: G70A tRNA minihelix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,2336
Polymers64,5852
Non-polymers6484
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4020 Å2
ΔGint-40 kcal/mol
Surface area27170 Å2
MethodPISA
2
C: CCA-adding enzyme
D: G70A tRNA minihelix
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,8269
Polymers64,5852
Non-polymers1,2417
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4970 Å2
ΔGint-64 kcal/mol
Surface area27640 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
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain C
12chain B
22chain D

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain AA1 - 437
211chain CC1 - 443
112chain BB1 - 50
212chain DD1 - 50

NCS ensembles :
ID
1
2

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Components

#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 G70A tRNA minihelix


Mass: 10912.527 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H16N3O14P3
#4: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mn
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / 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: 2.79 Å3/Da / Density % sol: 55.88 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / pH: 5.6
Details: 18.6% PEG 3,350, 0.2M potassium sodium tartrate, 0.1M Sodium Acetate pH 5.6
PH range: 5.6

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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: Oct 3, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 3.201→29.791 Å / Num. obs: 23753 / % possible obs: 99.2 % / Redundancy: 3.2 % / Biso Wilson estimate: 67.73 Å2 / Rpim(I) all: 0.075 / Rrim(I) all: 0.139 / Rsym value: 0.116 / Net I/av σ(I): 6.214 / Net I/σ(I): 8.4 / Num. measured all: 75872
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRsym valueNet I/σ(I) obs% possible all
3.2-3.373.20.4831.61088733890.3130.4832.598.7
3.37-3.583.20.3172.51043632370.2040.3173.799.4
3.58-3.833.30.2123.51008830500.1360.2125.599.6
3.83-4.133.20.1445.3897628360.0930.1447.299.3
4.13-4.533.20.1047.2839926190.0670.1049.599.4
4.53-5.063.30.0838.8783223970.0530.08311.699.5
5.06-5.843.10.0799675921520.0510.07911.499.5
5.84-7.163.20.0769.3576618190.0490.07612.199.5
7.16-10.1230.05311.8437714430.0350.05317.199.4
10.12-29.7912.90.0431323528110.030.0432195.2

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
SCALA3.3.20data scaling
XDSdata reduction
PDB_EXTRACT3.14data extraction
PHASERphasing
PHENIX(phenix.refine: 1.8.2_1309)refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.201→29.68 Å / SU ML: 0.47 / Cross valid method: FREE R-VALUE / σ(F): 1.11 / Phase error: 26.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2511 2178 5.11 %
Rwork0.2095 40409 -
obs0.2116 -95.01 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 237.18 Å2 / Biso mean: 83.4007 Å2 / Biso min: 24.59 Å2
Refinement stepCycle: final / Resolution: 3.201→29.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7297 1452 130 2 8881
Biso mean--95.71 26.97 -
Num. residues----948
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0049180
X-RAY DIFFRACTIONf_angle_d0.75512706
X-RAY DIFFRACTIONf_chiral_restr0.031395
X-RAY DIFFRACTIONf_plane_restr0.0041377
X-RAY DIFFRACTIONf_dihedral_angle_d13.2773702
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4105X-RAY DIFFRACTION9.157TORSIONAL
12C4105X-RAY DIFFRACTION9.157TORSIONAL
21B838X-RAY DIFFRACTION9.157TORSIONAL
22D838X-RAY DIFFRACTION9.157TORSIONAL
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 (%)
3.2003-3.26980.38231180.33622303242186
3.2698-3.34580.35561360.30612518265496
3.3458-3.42930.35021140.27712575268995
3.4293-3.52190.32591570.27142514267196
3.5219-3.62540.35131370.25822524266195
3.6254-3.74220.30631460.24242545269196
3.7422-3.87570.2611000.23912616271696
3.8757-4.03050.24191430.21192473261694
4.0305-4.21340.25691000.20362582268295
4.2134-4.43490.25431400.19592515265595
4.4349-4.71170.24711420.17922528267096
4.7117-5.07380.25391420.17882561270395
5.0738-5.58140.23261570.18612511266896
5.5814-6.3820.23641560.20812558271496
6.382-8.01440.19891630.17692516267996
8.0144-29.68130.16011270.15692570269796

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