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- EMDB-1632: Structural analysis of substrate binding by the TatBC component o... -

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Basic information

Entry
Database: EMDB / ID: EMD-1632
TitleStructural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
Map dataThis is a 3D map of a small TatBC complex.
Sample
  • Sample: tatBC small structure
  • Protein or peptide: twin arginine transporter
Keywordstwin arginine / Tat / protein transport / blue native PAGE / single particle electron microscopy
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining
AuthorsTarry MJ / Schaefer E / Chen S / Buchanan G / Greene NP / Lea SM / Palmer T / Saibil HR / Berks BC
CitationJournal: Proc Natl Acad Sci U S A / Year: 2009
Title: Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system.
Authors: Michael J Tarry / Eva Schäfer / Shuyun Chen / Grant Buchanan / Nicholas P Greene / Susan M Lea / Tracy Palmer / Helen R Saibil / Ben C Berks /
Abstract: The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the ...The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.
History
DepositionJul 8, 2009-
Header (metadata) releaseJul 21, 2009-
Map releaseJul 21, 2009-
UpdateOct 10, 2012-
Current statusOct 10, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 900
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 900
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1632.gif

Downloads & links

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Map

FileDownload / File: emd_1632.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D map of a small TatBC complex.
Voxel sizeX=Y=Z: 5 Å
Density
Contour LevelBy AUTHOR: 920.0 / Movie #1: 900
Minimum - Maximum-4484.300000000000182 - 3683.539999999999964
Average (Standard dev.)-48.429699999999997 (±366.742999999999995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 320 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z555
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-4484.3053683.536-48.430

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Supplemental data

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Sample components

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Entire : tatBC small structure

EntireName: tatBC small structure
Components
  • Sample: tatBC small structure
  • Protein or peptide: twin arginine transporter

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Supramolecule #1000: tatBC small structure

SupramoleculeName: tatBC small structure / type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: twin arginine transporter

MacromoleculeName: twin arginine transporter / type: protein_or_peptide / ID: 1 / Name.synonym: tat / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)

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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

Concentration0.014 mg/mL
StainingType: NEGATIVE
Details: negatively stained with 2% (wt/vol) uranyl acetate on glow discharged, continuous carbon-coated 300 mesh copper grids (Agar Scientific).
GridDetails: 300
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
Sample stageSpecimen holder: eucentric / Specimen holder model: OTHER / Tilt angle max: 50
DetailsElectron micrographs were recorded with low dose
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm
Details: Micrographs were digitized on a Zeiss SCAI scanner at a pixel size of 7 microns, corresponding to 1.667 angstroms on the specimen. Subsequently, adjacent pixels were 3 x 3 averaged to yield ...Details: Micrographs were digitized on a Zeiss SCAI scanner at a pixel size of 7 microns, corresponding to 1.667 angstroms on the specimen. Subsequently, adjacent pixels were 3 x 3 averaged to yield a pixel size of 5 angstroms.
Tilt angle min0

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Image processing

CTF correctionDetails: phase flipping
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Software - Name: SPIDER
Details: The tilted images were corrected for the effects of the contrast transfer function (CTF) by phase flipping, taking into account the defocus gradient across the micrographs and the position ...Details: The tilted images were corrected for the effects of the contrast transfer function (CTF) by phase flipping, taking into account the defocus gradient across the micrographs and the position of each particle. Images were processed using SPIDER version 11.12 and 15.06. Three-dimensional reconstruction was performed by the random conical tilt method in SPIDER. The particles were windowed into 64 x 64 pixel boxes.
Number images used: 1358

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