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- EMDB-44472: Alkalihalobacillus halodurans (Aha) trp RNA binding attenuation p... -

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Basic information

Entry
Database: EMDB / ID: EMD-44472
TitleAlkalihalobacillus halodurans (Aha) trp RNA binding attenuation protein (TRAP) mutant dTRAP without Trp
Map data
Sample
  • Complex: Hexamer dTRAP protein complex without Trp
    • Protein or peptide: Transcription attenuation protein MtrB
KeywordsHexamer of dTRAP apo / RNA BINDING PROTEIN
Function / homologyTranscription attenuation protein MtrB / Tryptophan RNA-binding attenuator protein domain / Tryptophan RNA-binding attenuator protein / Tryptophan RNA-binding attenuator protein-like domain superfamily / DNA-templated transcription termination / regulation of DNA-templated transcription / RNA binding / Transcription attenuation protein MtrB
Function and homology information
Biological speciesHalalkalibacterium halodurans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.24 Å
AuthorsYang H / Stachowski K / Foster M
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM077234 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM062750 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM120923 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128577 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM.
Authors: Weicheng Li / Haoyun Yang / Kye Stachowski / Andrew S Norris / Katie Lichtenthal / Skyler Kelly / Paul Gollnick / Vicki H Wysocki / Mark P Foster /
Abstract: The homo-dodecameric ring-shaped RNA binding attenuation protein (TRAP) from binds up to twelve tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of ...The homo-dodecameric ring-shaped RNA binding attenuation protein (TRAP) from binds up to twelve tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the operon mRNA, thereby downregulating biosynthesis of Trp. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity in TRAP, we engineered variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated the binding sites of alternating protomers such that only every other site was competent for Trp binding (WT-Mut dTRAP). Ligand binding monitored by NMR, calorimetry, and native mass spectrometry revealed strong cooperativity in dTRAP containing adjacent binding-competent sites, but a severe binding defect when the wild-type sites were separated by mutated sites. Cryo-EM experiments of dTRAP in its ligand-free apo state, and both dTRAP and WT-Mut dTRAP in the presence of Trp, revealed progressive stabilization of loops that gate the Trp binding site and participate in RNA binding. These studies provide important insights into the thermodynamic and structural basis for the observed ligand binding cooperativity in TRAP. Such insights can be useful for understanding allosteric control networks and for the development of those with defined ligand sensitivity and regulatory control.
History
DepositionApr 15, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44472.png

Downloads & links

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Map

FileDownload / File: emd_44472.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 256 pix.
= 219.648 Å		0.86 Å/pix.
x 256 pix.
= 219.648 Å		0.86 Å/pix.
x 256 pix.
= 219.648 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.858 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.135
Minimum - Maximum-0.3665253 - 0.5110273
Average (Standard dev.)0.0007831318 (±0.016348941)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 219.648 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44472_half_map_1.map
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Half map: #2

Fileemd_44472_half_map_2.map
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Sample components

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Entire : Hexamer dTRAP protein complex without Trp

EntireName: Hexamer dTRAP protein complex without Trp
Components
  • Complex: Hexamer dTRAP protein complex without Trp
    • Protein or peptide: Transcription attenuation protein MtrB

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Supramolecule #1: Hexamer dTRAP protein complex without Trp

SupramoleculeName: Hexamer dTRAP protein complex without Trp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Halalkalibacterium halodurans (bacteria)

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Macromolecule #1: Transcription attenuation protein MtrB

MacromoleculeName: Transcription attenuation protein MtrB / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Halalkalibacterium halodurans (bacteria)
Molecular weightTheoretical: 18.418471 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MNVGDNSNFF VIKAKENGVN VFGMTRGTDT RFHHSEKLDK GEVMIAQFTE HTSAVKIRGK AIIQTSYGTL DTEKDEGGGG SGGGGSMNV GDNSNFFVIK AKENGVNVFG MTRGTDTRFH HSEKLDKGEV MIAQFTEHTS AVKIRGKAII QTSYGTLDTE K DEENLYFQ

UniProtKB: Transcription attenuation protein MtrB

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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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium Chloride

Details: 20 mM HEPES, 200 mM NaCl, pH 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
Details: 20 mA, 30 sec hold, 1 min glow discharge using a PELCO easiGlow Discharge System
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: lotted with a blot force of 1 for 4 seconds at 277.15K and 100% relative humidity before being plunged frozen into liquid ethane.
DetailsAn aliquot of 10 mg/mL of apo dTRAP was diluted with cryo-EM buffer (20 mM HEPES, 200 mM NaCl, pH 8) supplemented with a 0.6% stock of Triton X-100 to obtain a final sample of 6 mg/mL protein and 0.05% Triton X-100.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91038
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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