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- EMDB-2529: Single particle electron microscopy of the human mitochondrial tr... -

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Basic information

Entry
Database: EMDB / ID: EMD-2529
TitleSingle particle electron microscopy of the human mitochondrial transcription initiation complex
Map dataReconstruction of the human mitochondrial transcription initiation complex
Sample
  • Sample: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
  • Protein or peptide: Dimethyladenosine transferase 2, mitochondrial
  • Protein or peptide: Dimethyladenosine transferase 2, mitochondrial
  • Protein or peptide: Transcription factor A, mitochondrial
Keywordshuman mitochondrial transcription / POLRMT / TFAM / TFB2M
Function / homology
Function and homology information


Mitochondrial transcription initiation / mitochondrial promoter sequence-specific DNA binding / mitochondrial respiratory chain complex assembly / mitochondrial transcription factor activity / transcription initiation at mitochondrial promoter / rRNA (adenine-N6,N6-)-dimethyltransferase activity / mitochondrial transcription / rRNA methylation / mitochondrial nucleoid / heat shock protein binding ...Mitochondrial transcription initiation / mitochondrial promoter sequence-specific DNA binding / mitochondrial respiratory chain complex assembly / mitochondrial transcription factor activity / transcription initiation at mitochondrial promoter / rRNA (adenine-N6,N6-)-dimethyltransferase activity / mitochondrial transcription / rRNA methylation / mitochondrial nucleoid / heat shock protein binding / Transferases; Transferring one-carbon groups; Methyltransferases / response to nutrient / Transcriptional activation of mitochondrial biogenesis / transcription coactivator binding / sequence-specific DNA binding / response to hypoxia / transcription cis-regulatory region binding / mitochondrial matrix / chromatin binding / positive regulation of DNA-templated transcription / protein-containing complex / mitochondrion / RNA binding / nucleus / cytosol
Similarity search - Function
Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / HMG-box domain / HMG (high mobility group) box / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain ...Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / HMG-box domain / HMG (high mobility group) box / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
Transcription factor A, mitochondrial / Dimethyladenosine transferase 2, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsYakubovskaya E / Guja KE / Eng ET / Choi WS / Mejia E / Beglov B / Lukin M / Kozakov D / Garcia-Diaz M
CitationJournal: Nucleic Acids Res / Year: 2014
Title: Organization of the human mitochondrial transcription initiation complex.
Authors: Elena Yakubovskaya / Kip E Guja / Edward T Eng / Woo Suk Choi / Edison Mejia / Dmitri Beglov / Mark Lukin / Dima Kozakov / Miguel Garcia-Diaz /
Abstract: Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human ...Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein-protein interactions might help mediate initiation. We demonstrate that, in the absence of DNA, only POLRMT and TFAM form a stable interaction, albeit one with low affinity. This is consistent with the expected transient nature of the interactions necessary for initiation and implies that the promoter DNA acts as a scaffold that enables formation of the full initiation complex. Docking of known crystal structures into our EM maps results in a model for transcriptional initiation that strongly correlates with new and existing biochemical observations. Our results reveal the organization of TFAM, POLRMT and TFB2M around the LSP and represent the first structural characterization of the entire mitochondrial transcriptional initiation complex.
History
DepositionDec 6, 2013-
Header (metadata) releaseJan 15, 2014-
Map releaseJan 29, 2014-
UpdateApr 16, 2014-
Current statusApr 16, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.61
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.61
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2529.png

Downloads & links

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Map

FileDownload / File: emd_2529.map.gz / Format: CCP4 / Size: 284.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the human mitochondrial transcription initiation complex
Voxel sizeX=Y=Z: 3.4 Å
Density
Contour LevelBy EMDB: 4.77 / Movie #1: 4.61
Minimum - Maximum-2.27669072 - 6.27684975
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-6-6-6
Dimensions424242
Spacing424242
CellA=B=C: 142.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.43.43.4
M x/y/z424242
origin x/y/z0.0000.0000.000
length x/y/z142.800142.800142.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-6-6-6
NC/NR/NS424242
D min/max/mean-2.2776.277-0.000

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Supplemental data

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Sample components

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Entire : Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA

EntireName: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
Components
  • Sample: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
  • Protein or peptide: Dimethyladenosine transferase 2, mitochondrial
  • Protein or peptide: Dimethyladenosine transferase 2, mitochondrial
  • Protein or peptide: Transcription factor A, mitochondrial

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Supramolecule #1000: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA

SupramoleculeName: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA / type: sample / ID: 1000
Details: The complex also has a DNA, that has a mitochondrial light strand promoter sequence
Oligomeric state: one POLRMT one TFB2M and one TFAM TFAM) / Number unique components: 3
Molecular weightExperimental: 250 KDa / Theoretical: 250 KDa / Method: Gel filtration, electrophoresis

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Macromolecule #1: Dimethyladenosine transferase 2, mitochondrial

MacromoleculeName: Dimethyladenosine transferase 2, mitochondrial / type: protein_or_peptide / ID: 1 / Name.synonym: TFB2M / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 39 KDa / Theoretical: 39 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Dimethyladenosine transferase 2, mitochondrial

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Macromolecule #2: Dimethyladenosine transferase 2, mitochondrial

MacromoleculeName: Dimethyladenosine transferase 2, mitochondrial / type: protein_or_peptide / ID: 2 / Name.synonym: TFB2M / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 39 KDa / Theoretical: 39 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Dimethyladenosine transferase 2, mitochondrial

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Macromolecule #3: Transcription factor A, mitochondrial

MacromoleculeName: Transcription factor A, mitochondrial / type: protein_or_peptide / ID: 3 / Name.synonym: TFAM / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightExperimental: 29 KDa / Theoretical: 29 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Transcription factor A, mitochondrial

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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

Concentration0.2 mg/mL
BufferpH: 8 / Details: 50mM KCl,20mM,HEPES, 10mM MgCl2
StainingType: NEGATIVE
Details: Grids with adsorbed complex were washed 2 times in buffer and then floated on 1% w/v uranyl acetate for 30 seconds.
GridDetails: 200 mesh gold grid with thin carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 88249 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.5 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 50,000 times magnification at each new area of the grid
DateOct 21, 2013
Image recordingCategory: CCD / Film or detector model: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 200
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: EMAN2 protocol each particle
Final two d classificationNumber classes: 15
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 12453
DetailsReference free classes were divided into two subsets, based on particle size, and two initial models were generated from each subset. One model had a visibly elongated shape, whereas the other was more compact. We utilized both models as starting points for parallel refinement, as implemented in EMAN MULTIREFINE routine

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Atomic model buiding 1

Initial modelPDB ID:

3spa


Chain - Chain ID: A
SoftwareName: Chimera, SITUS
Details3 proteins were fitted separately by manual docking and then refine with SITUS and in-home program.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4gs5


Chain - Chain ID: A
SoftwareName: Chimera, SITUS
Details3 proteins were fitted separately by manual docking and then refine with SITUS and in-home program.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

3tq6


Chain - Chain ID: A
SoftwareName: Chimera, SITUS
Details3 proteins were fitted separately by manual docking and then refine with SITUS and in-home program.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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