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- EMDB-3450: Structure of the K+ transporter KtrAB from Vibrio alginolyticus i... -

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Basic information

Entry
Database: EMDB / ID: EMD-3450
TitleStructure of the K+ transporter KtrAB from Vibrio alginolyticus in the ADP-bound state
Map dataKtrB2-KtrA8-KtrB2 complex
Sample
  • Complex: KtrAB
    • Protein or peptide: KtrB
    • Protein or peptide: KtrA
Biological speciesVibrio alginolyticus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsDiskowski M / Mills DJ / Baerland N / Haenelt I / Vonck J
CitationJournal: Elife / Year: 2017
Title: Helical jackknives control the gates of the double-pore K uptake system KtrAB.
Authors: Marina Diskowski / Ahmad Reza Mehdipour / Dorith Wunnicke / Deryck J Mills / Vedrana Mikusevic / Natalie Bärland / Jan Hoffmann / Nina Morgner / Heinz-Jürgen Steinhoff / Gerhard Hummer / ...Authors: Marina Diskowski / Ahmad Reza Mehdipour / Dorith Wunnicke / Deryck J Mills / Vedrana Mikusevic / Natalie Bärland / Jan Hoffmann / Nina Morgner / Heinz-Jürgen Steinhoff / Gerhard Hummer / Janet Vonck / Inga Hänelt /
Abstract: Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K channels, RCK domains regulate ion fluxes. Until now, a single ...Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from , determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.
History
DepositionOct 25, 2016-
Header (metadata) releaseNov 23, 2016-
Map releaseMay 31, 2017-
UpdateMar 28, 2018-
Current statusMar 28, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0552
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0552
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3450.png

Downloads & links

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Map

FileDownload / File: emd_3450.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKtrB2-KtrA8-KtrB2 complex
Voxel sizeX=Y=Z: 1.67 Å
Density
Contour LevelBy AUTHOR: 0.0552 / Movie #1: 0.0552
Minimum - Maximum-0.04200034 - 0.12103695
Average (Standard dev.)0.0011452492 (±0.007234686)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.671.671.67
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z320.640320.640320.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0420.1210.001

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Supplemental data

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Sample components

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Entire : KtrAB

EntireName: KtrAB
Components
  • Complex: KtrAB
    • Protein or peptide: KtrB
    • Protein or peptide: KtrA

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Supramolecule #1: KtrAB

SupramoleculeName: KtrAB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: complex of KtrB2A8 with another KtrB transporter dimer attached to the symmetric A8 regulatory domain
Source (natural)Organism: Vibrio alginolyticus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pBAD18
Molecular weightTheoretical: 340 KDa

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Macromolecule #1: KtrB

MacromoleculeName: KtrB / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio alginolyticus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQFHQRGVF YVPDGKRDKA KGGEPRIILL SFLGVLLPSA VLLTLPVFSV SGLSITDALF TATSAISVT GLGVVDTGQH FTLAGKILLM CLMQIGGLGQ MTLSAVLLYM FGVRLSLRQQ A LAKEALGQ ERQVNLRRLV KKIVTFALVA EAIGFVFLSY RWVPEMGWQT ...String:
MTQFHQRGVF YVPDGKRDKA KGGEPRIILL SFLGVLLPSA VLLTLPVFSV SGLSITDALF TATSAISVT GLGVVDTGQH FTLAGKILLM CLMQIGGLGQ MTLSAVLLYM FGVRLSLRQQ A LAKEALGQ ERQVNLRRLV KKIVTFALVA EAIGFVFLSY RWVPEMGWQT GMFYALFHSI SA FNNAGFA LFSDSMMSFV NDPLVSFTLA GLFIFGGLGF TVIGDVWRHW RKGFHFLHIH TKI MLIATP LLLLVGTVLF WLLERHNPNT MGSLTTGGQW LAAFFQSASA RTAGFNSVDL TQFT QPALL IMIVLMLIGA GSTSTGGGIK VSTFAVAFMA TWTFLRQKKH VVMFKRTVNW PTVTK SLAI IVVSGAILTT AMFLLMLTEK ASFDKVMFET ISAFATVGLT AGLTAELSEP GKYIMI VVM IIGRIGPLTL AYMLARPEPT LIKYPEDTVL TG

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Macromolecule #2: KtrA

MacromoleculeName: KtrA / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vibrio alginolyticus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKTGDKQFAV IGLGRFGLAV CKELQDSGSQ VLAVDINEDR VKEAAGFVSQ AIVANCTHEE TVAELKLDD YDMVMIAIGA DVNASILATL IAKEAGVKSV WVKANDRFQA RVLQKIGADH I IMPERDMG IRVARKMLDK RVLEFHPLGS GLAMTEFVVG SRLMGKTLSD ...String:
MKTGDKQFAV IGLGRFGLAV CKELQDSGSQ VLAVDINEDR VKEAAGFVSQ AIVANCTHEE TVAELKLDD YDMVMIAIGA DVNASILATL IAKEAGVKSV WVKANDRFQA RVLQKIGADH I IMPERDMG IRVARKMLDK RVLEFHPLGS GLAMTEFVVG SRLMGKTLSD LALCKVEGVQ VL GYKRGPE IIKAPDMSTT LEIGDLIIVV GPQDKLANKL KSL

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris/HCl
70.0 mMNaClSodium chlorideNaClSodium chloride
30.0 mMKCl
1.5 mMCymal-6
0.6 mMADPAdenosine diphosphate
GridModel: C-flat CF-MH-4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: The sample was blotted for 10 seconds.

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 30675 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 20000
Specialist opticsEnergy filter - Name: In-column Omega Filter
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-40 / Number real images: 800 / Average exposure time: 8.0 sec. / Average electron dose: 27.0 e/Å2

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Image processing

Particle selectionNumber selected: 35400
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4j7c

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 3 / Avg.num./class: 12000 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 20500
FSC plot (resolution estimation)

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