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- PDB-5yki: Crystal structure of the engineered nine-repeat PUF domain in com... -

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Basic information

Entry
Database: PDB / ID: 5yki
TitleCrystal structure of the engineered nine-repeat PUF domain in complex with cognate 9nt-RNA
Components
  • Pumilio homolog 1
  • RNA (5'-R(*UP*GP*UP*UP*GP*UP*AP*UP*A)-3')
KeywordsRNA BINDING PROTEIN/RNA / PUF repeats / engineered protein / RNA recognition / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


regulation of miRNA-mediated gene silencing / post-transcriptional gene silencing / positive regulation of miRNA-mediated gene silencing / regulation of chromosome segregation / positive regulation of RIG-I signaling pathway / 3'-UTR-mediated mRNA destabilization / miRNA processing / miRNA binding / post-transcriptional regulation of gene expression / Golgi Associated Vesicle Biogenesis ...regulation of miRNA-mediated gene silencing / post-transcriptional gene silencing / positive regulation of miRNA-mediated gene silencing / regulation of chromosome segregation / positive regulation of RIG-I signaling pathway / 3'-UTR-mediated mRNA destabilization / miRNA processing / miRNA binding / post-transcriptional regulation of gene expression / Golgi Associated Vesicle Biogenesis / mRNA destabilization / regulation of mRNA stability / mRNA 3'-UTR binding / adult locomotory behavior / stem cell differentiation / P-body / cytoplasmic stress granule / regulation of translation / spermatogenesis / regulation of cell cycle / axon / RNA binding / nucleoplasm / cytoplasm / cytosol
Similarity search - Function
Pumilio, RNA binding domain / Pumilio homology domain / Pumilio homology domain (PUM-HD) profile. / Pumilio-family RNA binding repeat / Pumilio RNA-binding repeat profile. / Pumilio RNA-binding repeat / Pumilio-like repeats / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
RNA / Pumilio homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.25 Å
AuthorsZhao, Y.Y. / Wang, J. / Li, H.T. / Wang, Z.X. / Wu, J.W.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2016YFA0502004 China
National Science Foundation (China)31621063 China
CitationJournal: Nucleic Acids Res. / Year: 2018
Title: Expanding RNA binding specificity and affinity of engineered PUF domains.
Authors: Zhao, Y.Y. / Mao, M.W. / Zhang, W.J. / Wang, J. / Li, H.T. / Yang, Y. / Wang, Z. / Wu, J.W.
History
DepositionOct 14, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 14, 2018Provider: repository / Type: Initial release
Revision 1.1Jun 6, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 22, 2023Group: 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

Downloads & links

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Assembly

Deposited unit
A: Pumilio homolog 1
C: RNA (5'-R(*UP*GP*UP*UP*GP*UP*AP*UP*A)-3')


Theoretical massNumber of molelcules
Total (without water)50,8972
Polymers50,8972
Non-polymers00
Water2,594144
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, Interaction of protein and RNA is determined by the fluorescence polarization assay.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2930 Å2
ΔGint-8 kcal/mol
Surface area19070 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.045, 70.318, 97.992
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Pumilio homolog 1 / Pumilio-1


Mass: 48061.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This is an engineered protein, which is made up of fragments of the RNA binding domain (PUF domain) of human Pumilio1 (Uniprot No. Q14671).
Source: (gene. exp.) Homo sapiens (human) / Gene: PUM1, KIAA0099, PUMH1 / Plasmid: pET-Duet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q14671
#2: RNA chain RNA (5'-R(*UP*GP*UP*UP*GP*UP*AP*UP*A)-3')


Mass: 2834.695 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 144 / 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: 2.3 Å3/Da / Density % sol: 46.59 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.8 / Details: 0.1M BIS-TRIS Propane, pH 8.8, 20% PEG MME 550

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.91939 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: May 24, 2013
RadiationMonochromator: double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91939 Å / Relative weight: 1
ReflectionResolution: 2.25→50 Å / Num. obs: 22954 / % possible obs: 99.6 % / Redundancy: 9.4 % / Biso Wilson estimate: 33.8 Å2 / Rmerge(I) obs: 0.12 / Net I/σ(I): 17.6
Reflection shellResolution: 2.25→2.29 Å / Redundancy: 7.5 % / Rmerge(I) obs: 0.501 / Mean I/σ(I) obs: 2.7 / Num. unique obs: 1095 / % possible all: 96.7

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
HKL-2000data collection
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1M8Z

1m8z


Resolution: 2.25→49 Å / SU ML: 0.23 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 22.61
RfactorNum. reflection% reflectionSelection details
Rfree0.236 1175 5.13 %Random selection
Rwork0.1968 ---
obs0.1988 22895 99.51 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 41.1 Å2
Refinement stepCycle: LAST / Resolution: 2.25→49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3114 187 0 144 3445
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0053380
X-RAY DIFFRACTIONf_angle_d0.6474598
X-RAY DIFFRACTIONf_dihedral_angle_d19.7052045
X-RAY DIFFRACTIONf_chiral_restr0.04515
X-RAY DIFFRACTIONf_plane_restr0.004567
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.25-2.350.30441390.24472604X-RAY DIFFRACTION97
2.3506-2.47460.27531450.2282662X-RAY DIFFRACTION100
2.4746-2.62960.27331540.23132689X-RAY DIFFRACTION100
2.6296-2.83260.23921540.21992684X-RAY DIFFRACTION100
2.8326-3.11760.26161560.22382697X-RAY DIFFRACTION100
3.1176-3.56860.26681500.20362725X-RAY DIFFRACTION100
3.5686-4.49560.20681350.16762771X-RAY DIFFRACTION100
4.4956-490.19181420.17462888X-RAY DIFFRACTION100

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