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- PDB-7zla: Cryo-EM structure of holo-PdxR from Bacillus clausii bound to its... -

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Basic information

Entry
Database: PDB / ID: 7zla
TitleCryo-EM structure of holo-PdxR from Bacillus clausii bound to its target DNA in the half-closed conformation
Components
  • (DNA (48-MER)) x 2
  • PLP-dependent aminotransferase family protein
KeywordsDNA BINDING PROTEIN / transcription factor / PLP-binding protein / domain-swap homodimer
Function / homology
Function and homology information


transaminase activity / biosynthetic process / pyridoxal phosphate binding / DNA-binding transcription factor activity
Similarity search - Function
GntR-type HTH domain profile. / helix_turn_helix gluconate operon transcriptional repressor / Transcription regulator HTH, GntR / Bacterial regulatory proteins, gntR family / Aminotransferase, class I/classII / Aminotransferase class I and II / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / PLP-dependent aminotransferase family protein
Similarity search - Component
Biological speciesAlkalihalobacillus clausii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.99 Å
AuthorsFreda, I. / Montemiglio, L.C. / Tramonti, A. / Contestabile, R. / Vallone, B. / Savino, C. / Exertier, C. / Bolognesi, M. / Chaves Sanjuan, A.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR.
Authors: Ida Freda / Cécile Exertier / Anna Barile / Antonio Chaves-Sanjuan / Mirella Vivoli Vega / Michail N Isupov / Nicholas J Harmer / Elena Gugole / Paolo Swuec / Martino Bolognesi / Anita ...Authors: Ida Freda / Cécile Exertier / Anna Barile / Antonio Chaves-Sanjuan / Mirella Vivoli Vega / Michail N Isupov / Nicholas J Harmer / Elena Gugole / Paolo Swuec / Martino Bolognesi / Anita Scipioni / Carmelinda Savino / Martino Luigi Di Salvo / Roberto Contestabile / Beatrice Vallone / Angela Tramonti / Linda Celeste Montemiglio /
Abstract: Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered ...Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5'-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process. Moreover, the resolution of an apo-PdxR crystallographic structure provided a detailed description of the transition of the effector domain to the holo-PdxR form triggered by the binding of the PLP effector molecule. Binding analyses of mutated DNA sequences using both wild type and PdxR variants revealed a central role of electrostatic interactions and of the intrinsic asymmetric bending of the DNA in allosterically guiding the holo-PdxR-DNA recognition process, from the first encounter through the fully bound state. Our results detail the structure and dynamics of the PdxR-DNA complex, clarifying the mechanism governing the DNA-binding mode of the holo-PdxR and the regulation features of the MocR family of transcription factors.
History
DepositionApr 14, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_3d_fitting_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PLP-dependent aminotransferase family protein
B: PLP-dependent aminotransferase family protein
C: DNA (48-MER)
D: DNA (48-MER)


Theoretical massNumber of molelcules
Total (without water)140,5604
Polymers140,5604
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13410 Å2
ΔGint-67 kcal/mol
Surface area51540 Å2
MethodPISA

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Components

#1: Protein PLP-dependent aminotransferase family protein


Mass: 55499.121 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alkalihalobacillus clausii (bacteria) / Gene: CHH72_17460 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A268NVG2
#2: DNA chain DNA (48-MER)


Mass: 14667.447 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Alkalihalobacillus clausii (bacteria)
#3: DNA chain DNA (48-MER)


Mass: 14894.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Alkalihalobacillus clausii (bacteria)
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Binary complex between the PLP-bound homodimeric PdxR and a 48bp double strand DNA fragment
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.136 MDa / Experimental value: NO
Source (natural)Organism: Alkalihalobacillus clausii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHepes1
250 mMNaClSodium chloride1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3284

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2.8image acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1358491
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68689 / Symmetry type: POINT
Atomic model buildingB value: 181 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7PQ9

7pq9


Accession code: 7PQ9 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 74.69 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059018
ELECTRON MICROSCOPYf_angle_d0.75112613
ELECTRON MICROSCOPYf_dihedral_angle_d24.4721853
ELECTRON MICROSCOPYf_chiral_restr0.0471383
ELECTRON MICROSCOPYf_plane_restr0.0061286

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