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- EMDB-5222: Nitrogen-Responsive Transcription Factor NrpR form Methanococcus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5222
TitleNitrogen-Responsive Transcription Factor NrpR form Methanococcus maripaludis in a 2OG-bound (inhibited) state
Map dataReconstruction volume of 2OG-bound NrpR from Methanococcus maripaludis
Sample
  • Sample: NrpR from Methanococcus maripaludis
  • Protein or peptide: NrpR
KeywordsNitrogen assimilation / Nitrogen regulation / Transcriptional regulation
Function / homologyNrpR regulatory domain
Function and homology information
Biological speciesMethanococcus maripaludis (archaea)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsWisedchaisri G / Dranow DM / Lie TJ / Bonanno JB / Patskovsky Y / Ozyurt SA / Sauder JM / Almo SC / Wasserman SR / Burley SK ...Wisedchaisri G / Dranow DM / Lie TJ / Bonanno JB / Patskovsky Y / Ozyurt SA / Sauder JM / Almo SC / Wasserman SR / Burley SK / Leigh JA / Gonen T
CitationJournal: Structure / Year: 2010
Title: Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR.
Authors: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John ...Authors: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John A Leigh / Tamir Gonen /
Abstract: Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. ...Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
History
DepositionAug 4, 2010-
Header (metadata) releaseNov 17, 2010-
Map releaseNov 17, 2010-
UpdateNov 17, 2010-
Current statusNov 17, 2010Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5222_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5222.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction volume of 2OG-bound NrpR from Methanococcus maripaludis
Voxel sizeX=Y=Z: 4.2 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-10.2882 - 11.349399999999999
Average (Standard dev.)-0.00000000132207 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-30-30
Dimensions606060
Spacing606060
CellA=B=C: 252 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-30-30-30
NC/NR/NS606060
D min/max/mean-10.28811.349-0.000

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Supplemental data

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Sample components

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Entire : NrpR from Methanococcus maripaludis

EntireName: NrpR from Methanococcus maripaludis
Components
  • Sample: NrpR from Methanococcus maripaludis
  • Protein or peptide: NrpR

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Supramolecule #1000: NrpR from Methanococcus maripaludis

SupramoleculeName: NrpR from Methanococcus maripaludis / type: sample / ID: 1000 / Oligomeric state: Dimer / Number unique components: 1
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa

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Macromolecule #1: NrpR

MacromoleculeName: NrpR / type: protein_or_peptide / ID: 1 / Name.synonym: MMP0607 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Methanococcus maripaludis (archaea) / Strain: Mm500RC / Location in cell: Cytoplasm
Molecular weightTheoretical: 600 KDa
SequenceInterPro: NrpR regulatory domain

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
Details: 100mM Tris HCl pH 7.5, 1M KCl and 5mM glycerol, 10mM 2-oxoglutarate
StainingType: NEGATIVE
Details: A 2 microlitre drop of NrpR was applied to a carbon-coated grid, washed 3 times with milliQ water and stained using 0.075% uranyl formate.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 52000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
Image recordingDigitization - Scanner: NIKON SUPER COOLSCAN 9000

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Details: Random conical tilt followed by angular refinement / Number images used: 8498

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Atomic model buiding 1

Initial modelPDB ID:

3nek


Chain - Chain ID: A
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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