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- PDB-6een: Crystal structure of a designer Pentatrico Peptide RNA binding pr... -

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Entry
Database: PDB / ID: 6een
TitleCrystal structure of a designer Pentatrico Peptide RNA binding protein, bound to a complex RNA target and featuring an infinite superhelix and microheterogeneity.
Components
  • (Designer Pentatricopeptide Protein ...) x 4
  • RNA (5'-R(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • RNA (5'-R(P*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
  • RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')
  • RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')
KeywordsRNA BINDING PROTEIN/RNA / Complex / PPR / RNA / Helical disorder / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA
Function and homology information
Biological speciesZea mays (maize)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.01 Å
AuthorsSchmidberger, J.W. / Bond, C.S.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC) Australia
CitationJournal: To Be Published
Title: Crystal structure of a designer Pentatrico Peptide RNA binding protein, bound to a complex RNA target and featuring an infinite superhelix and microheterogeneity.
Authors: Schmidberger, J.W. / Bond, C.S.
History
DepositionAug 15, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Oct 11, 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: Designer Pentatricopeptide Protein dPPR10
B: Designer Pentatricopeptide Protein dPPR10
C: Designer Pentatricopeptide Protein dPPR10
D: Designer Pentatricopeptide Protein dPPR10
F: RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')
G: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')
H: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
I: RNA (5'-R(P*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)151,1118
Polymers151,1118
Non-polymers00
Water3,855214
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, The protein is observed to bind to the RNA target. The actual target is GUAUCCUUAACCAUUUC. It was built as superposed poly X chains with occupancies ...Evidence: isothermal titration calorimetry, The protein is observed to bind to the RNA target. The actual target is GUAUCCUUAACCAUUUC. It was built as superposed poly X chains with occupancies matching their frequency in the actual sequence mentioned above.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)43.347, 51.647, 51.972
Angle α, β, γ (deg.)118.120, 97.210, 96.000
Int Tables number1
Space group name H-MP1
Detailsthis protein:RNA dimer assembly is represented as an octameric assembly due to structural disorder/microheterogeneity resulting in the structure being refined as 4 protein chains (partially occupied) + 4 RNA chains (partially occupied)

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Components

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Designer Pentatricopeptide Protein ... , 4 types, 4 molecules ABCD

#1: Protein Designer Pentatricopeptide Protein dPPR10


Mass: 34936.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zea mays (maize) / Production host: Escherichia coli (E. coli)
#2: Protein Designer Pentatricopeptide Protein dPPR10


Mass: 35053.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zea mays (maize) / Production host: Escherichia coli (E. coli)
#3: Protein Designer Pentatricopeptide Protein dPPR10


Mass: 34927.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zea mays (maize) / Production host: Escherichia coli (E. coli)
#4: Protein Designer Pentatricopeptide Protein dPPR10


Mass: 34801.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zea mays (maize) / Production host: Escherichia coli (E. coli)

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RNA chain , 4 types, 4 molecules FGHI

#5: RNA chain RNA (5'-R(P*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')


Mass: 3061.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Zea mays (maize)
#6: RNA chain RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')


Mass: 2710.535 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Zea mays (maize)
#7: RNA chain RNA (5'-R(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Mass: 2917.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Zea mays (maize)
#8: RNA chain RNA (5'-R(P*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')


Mass: 2701.679 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Zea mays (maize)

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Non-polymers , 1 types, 214 molecules

#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 214 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsThe structure features an infinite superhelix that has inherent helical disorder. This means we ...The structure features an infinite superhelix that has inherent helical disorder. This means we don't know where it begins and ends. This leads to map averaging at each position in the repeating helix. The highly redundant design of the protein means this averaging is not an issues except at positions 5 and 35 of each repeat that change to bind different bases in the target RNA. Also, the target RNA will average out. The solution to this was to build the structure as four superposed chains for the protein and four superposed chains for the RNA. Each case adding up to an occupancy of 1.
Sequence detailsProtein is designed based on a consensus sequence of many PPR proteins. Chains A to D are ...Protein is designed based on a consensus sequence of many PPR proteins. Chains A to D are repetitions of the same single chain with microheterogeneity at positions 5 and 35. The protein is observed to bind to the RNA target. The actual target sequence is GUAUCCUUAACCAUUUC. Because of infinite nature of superhelix, it was built as superposed poly X chains with occupancies matching their frequency in the actual sequence mentioned above.

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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: 2.73 Å3/Da / Density % sol: 54.97 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 0.1 M Mg Acetate, 0.05 M MES pH 5.6, 20% MPD

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.953737 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 1, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.953737 Å / Relative weight: 1
ReflectionResolution: 2.01→44.92 Å / Num. obs: 25127 / % possible obs: 97 % / Redundancy: 2 % / CC1/2: 0.995 / Rmerge(I) obs: 0.055 / Rpim(I) all: 0.049 / Rrim(I) all: 0.074 / Net I/σ(I): 9 / Num. measured all: 50507
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.01-2.061.60.305288417870.7860.2770.4132.190.8
8.98-44.921.90.0365442850.9850.0340.04924.299.7

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Processing

Software
NameVersionClassification
REFMACrefinement
Aimless0.5.31data scaling
PDB_EXTRACT3.24data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5i9f

5i9f


Resolution: 2.01→44.92 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.928 / WRfactor Rfree: 0.2435 / WRfactor Rwork: 0.177 / FOM work R set: 0.8049 / SU B: 17.923 / SU ML: 0.477 / SU Rfree: 0.3423 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.342 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2476 1207 4.8 %RANDOM
Rwork0.1832 ---
obs0.1862 23919 97.01 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 92.79 Å2 / Biso mean: 32.183 Å2 / Biso min: 11.54 Å2
Baniso -1Baniso -2Baniso -3
1-1.99 Å2-1.12 Å2-1.42 Å2
2---0.06 Å2-0.17 Å2
3----0.53 Å2
Refinement stepCycle: final / Resolution: 2.01→44.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9776 765 0 214 10755
Biso mean---35.37 -
Num. residues----1269
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.01410736
X-RAY DIFFRACTIONr_bond_other_d0.0010.01710111
X-RAY DIFFRACTIONr_angle_refined_deg1.6381.62914599
X-RAY DIFFRACTIONr_angle_other_deg1.2061.68823703
X-RAY DIFFRACTIONr_dihedral_angle_1_deg11.9535.2341283
X-RAY DIFFRACTIONr_dihedral_angle_2_deg41.55825.172435
X-RAY DIFFRACTIONr_dihedral_angle_3_deg20.49151979
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.0191536
X-RAY DIFFRACTIONr_chiral_restr0.0780.21422
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.0211807
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021769
LS refinement shellResolution: 2.008→2.06 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.34 60 -
Rwork0.295 1719 -
all-1779 -
obs--90.58 %

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