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- PDB-9be7: Alkalihalobacillus halodurans (Aha) trp RNA binding attenuation p... -

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Basic information

Entry
Database: PDB / ID: 9be7
TitleAlkalihalobacillus halodurans (Aha) trp RNA binding attenuation protein (TRAP) mutant dTRAP without Trp
ComponentsTranscription attenuation protein MtrB
KeywordsRNA BINDING PROTEIN / Hexamer of dTRAP apo
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.24 Å
AuthorsYang, H. / Stachowski, K. / Foster, M.
Funding support United States, 4items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transcription attenuation protein MtrB
D: Transcription attenuation protein MtrB
E: Transcription attenuation protein MtrB
F: Transcription attenuation protein MtrB
B: Transcription attenuation protein MtrB
C: Transcription attenuation protein MtrB


Theoretical massNumber of molelcules
Total (without water)110,5116
Polymers110,5116
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "C"
d_3ens_1chain "D"
d_4ens_1chain "A"
d_5ens_1chain "E"
d_6ens_1chain "F"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: ASN / Beg label comp-ID: ASN / End auth comp-ID: GLU / End label comp-ID: GLU / Auth seq-ID: 8 - 159 / Label seq-ID: 8 - 159

Dom-IDAuth asym-IDLabel asym-ID
d_1BE
d_2CF
d_3DB
d_4AA
d_5EC
d_6FD

NCS oper:
IDCodeMatrixVector
1given(0.499418023619, 0.866359760607, 0.00155012451041), (-0.866358045117, 0.499420224094, -0.00178253392537), (-0.00231847919522, -0.000452733270483, 0.99999720984)-40.3499182861, 150.327505177, 0.358952600183
2given(-0.999999966712, -0.000173272937621, -0.000191188571878), (0.000173339491188, -0.999999924375, -0.000348142672752), (-0.000191128233715, -0.000348175801693, 0.999999921122)219.693818718, 219.666010439, 0.0745286009641
3given(0.500260372553, -0.865874942674, -0.00037855170306), (0.865875014014, 0.500260247178, 0.00038105104346), (-0.00014056818187, -0.000518403198156, 0.999999855749)150.0172554, -40.24957273, 0.0860864622824
4given(-0.500969182365, -0.865449236573, 0.00524378069104), (0.865464649763, -0.500953380217, 0.00408054644329), (-0.000904616141854, 0.0065825348345, 0.999977925709)259.303431724, 69.3550645091, -0.679136708898
5given(-0.500271639482, 0.865868481763, 0.000242939131994), (-0.865868110557, -0.500271114163, -0.0011079054463), (-0.000837764976489, -0.000764606921213, 0.999999356763)69.6607463549, 259.978965274, 0.198719685065

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Components

#1: Protein
Transcription attenuation protein MtrB / Trp RNA-binding attenuation protein / TRAP / Tryptophan RNA-binding attenuator protein


Mass: 18418.471 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Halalkalibacterium halodurans (bacteria)
Gene: mtrB, BH1647 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9KCC6
Has protein modificationN

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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: Hexamer dTRAP protein complex without Trp / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Halalkalibacterium halodurans (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM HEPES, 200 mM NaCl, pH 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2200 mMSodium ChlorideNaCl1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: An 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 ...Details: An 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.
Specimen supportDetails: 20 mA, 30 sec hold, 1 min glow discharge using a PELCO easiGlow Discharge System
Grid material: GOLD / 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
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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91038 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 130.74 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00246438
ELECTRON MICROSCOPYf_angle_d0.44028616
ELECTRON MICROSCOPYf_chiral_restr0.0434972
ELECTRON MICROSCOPYf_plane_restr0.02821098
ELECTRON MICROSCOPYf_dihedral_angle_d11.4315840
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2EBELECTRON MICROSCOPYNCS constraints5.2395168416E-13
ens_1d_3EBELECTRON MICROSCOPYNCS constraints3.24618456266E-12
ens_1d_4EBELECTRON MICROSCOPYNCS constraints3.83945073577E-11
ens_1d_5EBELECTRON MICROSCOPYNCS constraints2.52850989225E-13
ens_1d_6EBELECTRON MICROSCOPYNCS constraints1.9552161319E-12

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