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- PDB-2i82: Crystal structure of pseudouridine synthase RluA: indirect sequen... -

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Basic information

Entry
Database: PDB / ID: 2i82
TitleCrystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure
Components
  • 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
  • Ribosomal large subunit pseudouridine synthase A
KeywordsLyase/RNA / pseudouridine synthase / Lyase-RNA COMPLEX
Function / homology
Function and homology information


tRNA pseudouridine32 synthase / 23S rRNA pseudouridine746 synthase / rRNA pseudouridine synthase activity / rRNA pseudouridine synthesis / enzyme-directed rRNA pseudouridine synthesis / RNA modification / tRNA pseudouridine synthase activity / pseudouridine synthesis / tRNA pseudouridine synthesis / pseudouridine synthase activity ...tRNA pseudouridine32 synthase / 23S rRNA pseudouridine746 synthase / rRNA pseudouridine synthase activity / rRNA pseudouridine synthesis / enzyme-directed rRNA pseudouridine synthesis / RNA modification / tRNA pseudouridine synthase activity / pseudouridine synthesis / tRNA pseudouridine synthesis / pseudouridine synthase activity / tRNA processing / rRNA processing / RNA binding
Similarity search - Function
Pseudouridine synthase, RluC/RluD / Pseudouridine synthase, RluA-like, conserved site / Rlu family of pseudouridine synthase signature. / Pseudouridine synthase, RsuA/RluA-like / RNA pseudouridylate synthase / Pseudouridine synthase / Pseudouridine synthase / Pseudouridine synthase, catalytic domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-FOU / RNA / RNA (> 10) / Dual-specificity RNA pseudouridine synthase RluA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.05 Å
AuthorsHoang, C.
CitationJournal: Mol.Cell / Year: 2006
Title: Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure
Authors: Hoang, C. / Chen, J. / Vizthum, C.A. / Kandel, J.M. / Hamilton, C.S. / Mueller, E.G. / Ferre-D'Amare, A.R.
History
DepositionAug 31, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 21, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
F: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
G: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
H: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
A: Ribosomal large subunit pseudouridine synthase A
B: Ribosomal large subunit pseudouridine synthase A
C: Ribosomal large subunit pseudouridine synthase A
D: Ribosomal large subunit pseudouridine synthase A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,20310
Polymers126,9028
Non-polymers3002
Water4,594255
1
E: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
A: Ribosomal large subunit pseudouridine synthase A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,8763
Polymers31,7262
Non-polymers1501
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
F: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
B: Ribosomal large subunit pseudouridine synthase A


Theoretical massNumber of molelcules
Total (without water)31,7262
Polymers31,7262
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
G: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
C: Ribosomal large subunit pseudouridine synthase A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,8763
Polymers31,7262
Non-polymers1501
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
H: 5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'
D: Ribosomal large subunit pseudouridine synthase A


Theoretical massNumber of molelcules
Total (without water)31,7262
Polymers31,7262
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)320.737, 51.691, 81.233
Angle α, β, γ (deg.)90.00, 90.81, 90.00
Int Tables number5
Space group name H-MC121
DetailsThe crystallographic model consists of all residues for the four RNA molecules in the asymmetric unit.

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Components

#1: RNA chain
5'-R(*GP*AP*GP*GP*GP*GP*AP*UP*UP*GP*AP*AP*AP*AP*UP*CP*CP*CP*CP*UP*C)-3'


Mass: 6642.013 Da / Num. of mol.: 4 / Source method: obtained synthetically
#2: Protein
Ribosomal large subunit pseudouridine synthase A / / rRNA-uridine lyase A / rRNA pseudouridylate synthase A


Mass: 25083.588 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rluA / Plasmid: pET15-b / Species (production host): Escherichia coli / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (DE3) / References: UniProt: P0AA37, pseudouridylate synthase
#3: Chemical ChemComp-FOU / (5S,6R)-5-FLUORO-6-HYDROXYDIHYDROPYRIMIDINE-2,4(1H,3H)-DIONE / (5S,6R)-5-FLUORO-6-HYDROXY-PSEUDOURIDINE


Mass: 150.108 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H7FN2O3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 255 / 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: 2

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Sample preparation

CrystalDensity Matthews: 2.64 Å3/Da / Density % sol: 53.45 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 25% PEG4000, 100mM Tris-HCl pH 8.5, 25mM MgCl2, 100mM LiCl, 1mM spermine, VAPOR DIFFUSION, HANGING DROP, temperature 295K
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG400011
2Tris-HClTris11
3MgCl211
4LiCl11
5spermine11
6H2O11
7PEG400012
8Tris-HClTris12
9MgCl212
10LiCl12
11spermine12

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.1
Wavelength: 0.9001, 0.92035, 0.92014, 0.942411, 0.979877, 0.979647
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jul 10, 2005
RadiationMonochromator: Double crystal, Si (111) / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.90011
20.920351
30.920141
40.9424111
50.9798771
60.9796471
ReflectionResolution: 2.05→19.83 Å / Num. obs: 82366 / % possible obs: 97.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 4.84 / Redundancy: 7.43 % / Biso Wilson estimate: 23.1 Å2 / Rmerge(I) obs: 0.069 / Rsym value: 0.069 / Net I/σ(I): 35.8
Reflection shellResolution: 2.05→2.18 Å / Redundancy: 6.96 % / Rmerge(I) obs: 0.467 / Mean I/σ(I) obs: 4.84 / Num. unique all: 8090 / Rsym value: 0.467 / % possible all: 97.2

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data collection
DENZOdata reduction
SCALEPACKdata scaling
SOLVEphasing
RefinementMethod to determine structure: MAD / Resolution: 2.05→19.83 Å / Rfactor Rfree error: 0.003 / Data cutoff high absF: 2402292.05 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.263 8255 10 %RANDOM
Rwork0.234 ---
all0.234 82366 --
obs0.234 82366 97.8 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 47.5999 Å2 / ksol: 0.344821 e/Å3
Displacement parametersBiso mean: 41.8 Å2
Baniso -1Baniso -2Baniso -3
1-1.76 Å20 Å24.04 Å2
2--2.85 Å20 Å2
3----4.61 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.34 Å0.29 Å
Luzzati d res low-5 Å
Luzzati sigma a0.32 Å0.24 Å
Refinement stepCycle: LAST / Resolution: 2.05→19.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6833 1746 20 255 8854
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.006
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_dihedral_angle_d24
X-RAY DIFFRACTIONc_improper_angle_d1.44
X-RAY DIFFRACTIONc_mcbond_it21.5
X-RAY DIFFRACTIONc_mcangle_it2.822
X-RAY DIFFRACTIONc_scbond_it2.912
X-RAY DIFFRACTIONc_scangle_it3.942.5
LS refinement shellResolution: 2.05→2.18 Å / Rfactor Rfree error: 0.009 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.333 1344 10 %
Rwork0.289 12153 -
obs-12153 97.2 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2dna-rna_rep.paramdna-rna.top
X-RAY DIFFRACTION3water_rep.paramwater.top
X-RAY DIFFRACTION4fou.parfou.top

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