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- PDB-5ykh: Crystal structure of the engineered nine-repeat PUF domain -

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Basic information

Entry
Database: PDB / ID: 5ykh
TitleCrystal structure of the engineered nine-repeat PUF domain
ComponentsPumilio homolog 1
KeywordsRNA BINDING PROTEIN / PUF repeats / engineered protein / RNA recognition
Function / homology
Function and homology information


regulation of miRNA-mediated gene silencing / positive regulation of miRNA-mediated gene silencing / regulation of chromosome segregation / positive regulation of RIG-I signaling pathway / post-transcriptional gene silencing / 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 / positive regulation of miRNA-mediated gene silencing / regulation of chromosome segregation / positive regulation of RIG-I signaling pathway / post-transcriptional gene silencing / 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 / adult locomotory behavior / mRNA 3'-UTR binding / stem cell differentiation / P-body / cytoplasmic stress granule / regulation of translation / spermatogenesis / regulation of cell cycle / axon / RNA binding / nucleoplasm / cytosol / cytoplasm
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
PHOSPHATE ION / Pumilio homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.457 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

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Assembly

Deposited unit
A: Pumilio homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,3474
Polymers48,0621
Non-polymers2853
Water1,27971
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The protein is monomeric in solution.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area210 Å2
ΔGint-5 kcal/mol
Surface area18900 Å2
MethodPISA
Unit cell
Length a, b, c (Å)203.052, 32.935, 61.364
Angle α, β, γ (deg.)90.00, 105.31, 90.00
Int Tables number5
Space group name H-MC121

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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: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 71 / 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: 2.06 Å3/Da / Density % sol: 40.26 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.5 / Details: 20% PEG 3350, 0.25M NaH2PO4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.97915 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 5, 2012
RadiationMonochromator: double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 2.45→50 Å / Num. obs: 14533 / % possible obs: 98.5 % / Redundancy: 6.9 % / Biso Wilson estimate: 41.75 Å2 / Rmerge(I) obs: 0.106 / Rpim(I) all: 0.045 / Rrim(I) all: 0.115 / Net I/σ(I): 14.9
Reflection shellResolution: 2.45→2.49 Å / Redundancy: 7.3 % / Rmerge(I) obs: 0.461 / Mean I/σ(I) obs: 8.2 / Num. unique obs: 745 / Rpim(I) all: 0.181 / Rrim(I) all: 0.495 / % possible all: 100

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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: 2.457→33.617 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.61
RfactorNum. reflection% reflectionSelection details
Rfree0.2584 715 4.93 %Random selection
Rwork0.2133 ---
obs0.2155 14491 98.21 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 46.5 Å2
Refinement stepCycle: LAST / Resolution: 2.457→33.617 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3098 0 15 71 3184
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0033166
X-RAY DIFFRACTIONf_angle_d0.4354266
X-RAY DIFFRACTIONf_dihedral_angle_d16.8481931
X-RAY DIFFRACTIONf_chiral_restr0.035469
X-RAY DIFFRACTIONf_plane_restr0.003551
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4569-2.64650.34391380.25372749X-RAY DIFFRACTION100
2.6465-2.91270.35361390.26732747X-RAY DIFFRACTION98
2.9127-3.33390.29211420.24772779X-RAY DIFFRACTION100
3.3339-4.19910.23191670.19782641X-RAY DIFFRACTION96
4.1991-33.61990.21451290.18532860X-RAY DIFFRACTION97

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