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- EMDB-14960: Cryo-EM structure of holo-PdxR from Bacillus clausii bound to its... -

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Basic information

Entry
Database: EMDB / ID: EMD-14960
TitleCryo-EM structure of holo-PdxR from Bacillus clausii bound to its target DNA in the open conformation
Map dataFull cryo-EM map of PdxR from Bacillus clausii in complex with its target DNA in the open conformation
Sample
  • Complex: Binary complex between the PLP-bound homodimeric PdxR and a 48bp double strand DNA fragment
    • Complex: PLP-dependent aminotransferase family protein
      • Protein or peptide: PLP-dependent aminotransferase family protein
    • Complex: DNA
      • DNA: DNA (48-MER)
      • DNA: DNA (48-MER)
Keywordstranscription factor / PLP-binding protein / domain-swap homodimer / DNA binding protein
Function / homology
Function and homology information


transaminase activity / biosynthetic process / pyridoxal phosphate binding / DNA-binding transcription factor activity / DNA binding
Similarity search - Function
: / Transcription regulator HTH, GntR / Bacterial regulatory proteins, gntR family / GntR-type HTH domain profile. / helix_turn_helix gluconate operon transcriptional repressor / 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
PLP-dependent aminotransferase family protein
Similarity search - Component
Biological speciesAlkalihalobacillus clausii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsFreda I / Montemiglio LC / Tramonti A / Contestabile R / Vallone B / Exertier C / Savino C / Chaves Sanjuan A / Bolognesi M
Funding support1 items
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
DepositionMay 10, 2022-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14960.png

Downloads & links

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Map

FileDownload / File: emd_14960.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull cryo-EM map of PdxR from Bacillus clausii in complex with its target DNA in the open conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 256 pix.
= 227.584 Å		0.89 Å/pix.
x 256 pix.
= 227.584 Å		0.89 Å/pix.
x 256 pix.
= 227.584 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.889 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.13
Minimum - Maximum-18.933005999999999 - 34.881059999999998
Average (Standard dev.)0.000000000015766 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 227.584 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half cryo-EM map 1 of PdxR from Bacillus...

Fileemd_14960_half_map_1.map
AnnotationHalf cryo-EM map 1 of PdxR from Bacillus clausii in complex with its target DNA in the open conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half cryo-EM map 2 of PdxR from Bacillus...

Fileemd_14960_half_map_2.map
AnnotationHalf cryo-EM map 2 of PdxR from Bacillus clausii in complex with its target DNA in the open conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Binary complex between the PLP-bound homodimeric PdxR and a 48bp ...

EntireName: Binary complex between the PLP-bound homodimeric PdxR and a 48bp double strand DNA fragment
Components
  • Complex: Binary complex between the PLP-bound homodimeric PdxR and a 48bp double strand DNA fragment
    • Complex: PLP-dependent aminotransferase family protein
      • Protein or peptide: PLP-dependent aminotransferase family protein
    • Complex: DNA
      • DNA: DNA (48-MER)
      • DNA: DNA (48-MER)

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Supramolecule #1: Binary complex between the PLP-bound homodimeric PdxR and a 48bp ...

SupramoleculeName: Binary complex between the PLP-bound homodimeric PdxR and a 48bp double strand DNA fragment
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 136 KDa

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Supramolecule #2: PLP-dependent aminotransferase family protein

SupramoleculeName: PLP-dependent aminotransferase family protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Alkalihalobacillus clausii (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Alkalihalobacillus clausii (bacteria)

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Macromolecule #1: PLP-dependent aminotransferase family protein

MacromoleculeName: PLP-dependent aminotransferase family protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Alkalihalobacillus clausii (bacteria)
Molecular weightTheoretical: 55.499121 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MELLWCELNR DLPTPLYEQL YAHIKTEITE GRIGYGTKLP SKRKLADSLK LSQNTVEAAY EQLVAEGYVE VIPRKGFYVQ AYEDLEYIR APQAPGDALA TKQDTIRYNF HPTHIDTTSF PFEQWRKYFK QTMCKENHRL LLNGDHQGEA SFRREIAYYL H HSRGVNCT ...String:
MELLWCELNR DLPTPLYEQL YAHIKTEITE GRIGYGTKLP SKRKLADSLK LSQNTVEAAY EQLVAEGYVE VIPRKGFYVQ AYEDLEYIR APQAPGDALA TKQDTIRYNF HPTHIDTTSF PFEQWRKYFK QTMCKENHRL LLNGDHQGEA SFRREIAYYL H HSRGVNCT PEQVVVGAGV ETLLQQLFLL LGESKVYGIE DPGYQLMRKL LSHYPNDYVP FQVDEEGIDV DSIVRTAVDV VY TTPSRHF PYGSVLSINR RKQLLHWAEA HENRYIIEDD YDSEFRYTGK TIPSLQSMDV HNKVIYLGAF S(LLP)SLIPSVR ISYMVLPAPL AHLYKNKFSY YHSTVSRIDQ QVLTAFMKQG DFEKHLNRMR KIYRRKLEKV LSLLKRYEDK LLIIGERSGL HIVLVVKNG MDEQTLVEKA LAAKAKVYPL SAYSLERAIH PPQIVLGFGS IPEDELEEAI ATVLNAWGFL VPRGSLEHHH H HH

UniProtKB: PLP-dependent aminotransferase family protein

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Macromolecule #2: DNA (48-MER)

MacromoleculeName: DNA (48-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Alkalihalobacillus clausii (bacteria)
Molecular weightTheoretical: 14.667447 KDa
SequenceString:
(DC)(DT)(DG)(DA)(DC)(DC)(DT)(DC)(DA)(DT) (DC)(DA)(DT)(DT)(DT)(DT)(DC)(DT)(DT)(DA) (DA)(DA)(DA)(DA)(DC)(DT)(DG)(DA)(DC) (DA)(DC)(DT)(DT)(DA)(DC)(DA)(DA)(DT)(DG) (DT) (DG)(DG)(DT)(DC)(DA)(DG)(DT)(DT)

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Macromolecule #3: DNA (48-MER)

MacromoleculeName: DNA (48-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Alkalihalobacillus clausii (bacteria)
Molecular weightTheoretical: 14.894607 KDa
SequenceString:
(DA)(DA)(DC)(DT)(DG)(DA)(DC)(DC)(DA)(DC) (DA)(DT)(DT)(DG)(DT)(DA)(DA)(DG)(DT)(DG) (DT)(DC)(DA)(DG)(DT)(DT)(DT)(DT)(DT) (DA)(DA)(DG)(DA)(DA)(DA)(DA)(DT)(DG)(DA) (DT) (DG)(DA)(DG)(DG)(DT)(DC)(DA)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5 / Component:
ConcentrationName
50.0 mMHepes
50.0 mMNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3284 / Average exposure time: 1.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1358491
Startup modelType of model: OTHER / Details: Initial 3D model generated in Relion 3.1
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 56199
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7pq9

source_name: PDB, initial_model_type: experimental model

7zla

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 151
Output model

PDB-7zth:
Cryo-EM structure of holo-PdxR from Bacillus clausii bound to its target DNA in the open conformation

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