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- PDB-6q52: Structure of a psychrophilic CCA-adding enzyme in complex with CM... -

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Basic information

Entry
Database: PDB / ID: 6q52
TitleStructure of a psychrophilic CCA-adding enzyme in complex with CMPcPP at room temperature in ChipX microfluidic device
ComponentsCCA-adding enzyme
KeywordsRNA BINDING PROTEIN / tRNA maturation / tRNA nucleotidyltransferase / ChipX / psychrophilic enzyme
Function / homology
Function and homology information


tRNA processing / nucleotidyltransferase activity / tRNA binding / nucleotide binding
Similarity search - Function
Serum Albumin; Chain A, Domain 1 - #80 / CCA-adding enzyme, C-terminal / tRNA nucleotidyltransferase domain 2 putative / : / tRNA nucleotidyltransferase/poly(A) polymerase, RNA and SrmB- binding domain / Probable RNA and SrmB- binding site of polymerase A / Poly A polymerase, head domain / Poly A polymerase head domain / Serum Albumin; Chain A, Domain 1 / Beta Polymerase, domain 2 ...Serum Albumin; Chain A, Domain 1 - #80 / CCA-adding enzyme, C-terminal / tRNA nucleotidyltransferase domain 2 putative / : / tRNA nucleotidyltransferase/poly(A) polymerase, RNA and SrmB- binding domain / Probable RNA and SrmB- binding site of polymerase A / Poly A polymerase, head domain / Poly A polymerase head domain / Serum Albumin; Chain A, Domain 1 / Beta Polymerase, domain 2 / Beta Polymerase; domain 2 / Nucleotidyltransferase superfamily / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chem-2TM / CCA-adding protein
Similarity search - Component
Biological speciesPlanococcus halocryophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
Authorsde Wijn, R. / Hennig, O. / Rollet, K. / Bluhm, A. / Betat, H. / Moerl, M. / Lorber, B. / Sauter, C.
Funding support France, 2items
OrganizationGrant numberCountry
French National Research AgencyANR-10-LABX-0036_NETRNA France
French National Research AgencyANR-11-LABX-0057_MITOCROSS France
CitationJournal: Iucrj / Year: 2019
Title: A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography.
Authors: de Wijn, R. / Hennig, O. / Roche, J. / Engilberge, S. / Rollet, K. / Fernandez-Millan, P. / Brillet, K. / Betat, H. / Morl, M. / Roussel, A. / Girard, E. / Mueller-Dieckmann, C. / Fox, G.C. ...Authors: de Wijn, R. / Hennig, O. / Roche, J. / Engilberge, S. / Rollet, K. / Fernandez-Millan, P. / Brillet, K. / Betat, H. / Morl, M. / Roussel, A. / Girard, E. / Mueller-Dieckmann, C. / Fox, G.C. / Olieric, V. / Gavira, J.A. / Lorber, B. / Sauter, C.
History
DepositionDec 6, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1May 29, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CCA-adding enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,9792
Polymers48,4981
Non-polymers4811
Water18010
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area18760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.450, 71.450, 293.610
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein CCA-adding enzyme


Mass: 48498.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planococcus halocryophilus (bacteria) / Gene: BBI08_05760 / Plasmid: pET-30b(+) / Details (production host): pET-30b Ek/LIC / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A1C7DQ98, CCA tRNA nucleotidyltransferase
#2: Chemical ChemComp-2TM / 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]methyl}phosphoryl]cytidine / CMPcPP


Mass: 481.184 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H18N3O13P3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / 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: 3.86 Å3/Da / Density % sol: 68.16 % / Description: bipyramid
Crystal growTemperature: 293 K / Method: counter-diffusion / pH: 7.5
Details: protein solution at 5.5 mg/mL in 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgCl2; reservoir solution: 30% (m/v) PEG3350, 200 mM ammonium chloride pH 6.6. Crystallization and crystallographic ...Details: protein solution at 5.5 mg/mL in 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgCl2; reservoir solution: 30% (m/v) PEG3350, 200 mM ammonium chloride pH 6.6. Crystallization and crystallographic analysis were performed using the ChipX microfluidic device.
PH range: 6.6-7.5

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Data collection

DiffractionMean temperature: 293 K / Ambient temp details: in situ / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 16, 2016
RadiationMonochromator: double-crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.3→47.77 Å / Num. obs: 34862 / % possible obs: 99.5 % / Redundancy: 6.7 % / Biso Wilson estimate: 60.56 Å2 / CC1/2: 0.987 / Rrim(I) all: 0.18 / Net I/σ(I): 6.9
Reflection shellResolution: 2.3→2.4 Å / Redundancy: 8.1 % / Num. unique obs: 4066 / CC1/2: 0.469 / Rrim(I) all: 2.312 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998)refinement
XDS20180808data reduction
XSCALE20180808data scaling
PHENIX(1.13_2998)phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1MIV

1miv


Resolution: 2.3→47.77 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 26.13
Details: triphosphate moiety was modelled in two conformations
RfactorNum. reflection% reflection
Rfree0.2293 1742 5 %
Rwork0.1997 --
obs0.2012 34840 99.49 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.3→47.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2989 0 29 10 3028
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0093107
X-RAY DIFFRACTIONf_angle_d0.9794205
X-RAY DIFFRACTIONf_dihedral_angle_d16.8791154
X-RAY DIFFRACTIONf_chiral_restr0.065469
X-RAY DIFFRACTIONf_plane_restr0.007519
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.36770.37111400.34942660X-RAY DIFFRACTION100
2.3677-2.44410.3651430.34282702X-RAY DIFFRACTION100
2.4441-2.53150.36211440.30812737X-RAY DIFFRACTION100
2.5315-2.63280.30451430.27372714X-RAY DIFFRACTION100
2.6328-2.75260.30311440.26992742X-RAY DIFFRACTION100
2.7526-2.89770.31641430.2692709X-RAY DIFFRACTION100
2.8977-3.07920.3181440.2422742X-RAY DIFFRACTION100
3.0792-3.31690.24911440.22392744X-RAY DIFFRACTION100
3.3169-3.65060.23481460.20692768X-RAY DIFFRACTION100
3.6506-4.17860.20931470.17192787X-RAY DIFFRACTION99
4.1786-5.26350.17641490.15122826X-RAY DIFFRACTION99
5.2635-47.78310.18551550.17152967X-RAY DIFFRACTION97

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