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- EMDB-20924: Single particle cryo-EM structure of KvAP -

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Basic information

Entry
Database: EMDB / ID: EMD-20924
TitleSingle particle cryo-EM structure of KvAP
Map dataKvAP-6E1 Fab complex
Sample
  • Complex: KvAP-6E1 Fab complex
    • Complex: KvAP
      • Protein or peptide: Voltage-gated potassium channel
    • Complex: 6E1 Fab
Keywordsvoltage-gated potassium channel / non-domain-swapped / TRANSPORT PROTEIN
Function / homologyVoltage-gated potassium channel / voltage-gated potassium channel activity / voltage-gated potassium channel complex / Ion transport domain / Ion transport protein / identical protein binding / Voltage-gated potassium channel
Function and homology information
Biological speciesAeropyrum pernix (archaea) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsTao X / MacKinnon R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM43949 United States
CitationJournal: Elife / Year: 2019
Title: Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology.
Authors: Xiao Tao / Roderick MacKinnon /
Abstract: Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two ...Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.
History
DepositionNov 5, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseDec 4, 2019-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.07
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 6.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uwm
  • Surface level: 6.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6uwm
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20924.png

Downloads & links

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Map

FileDownload / File: emd_20924.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKvAP-6E1 Fab complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 300 pix.
= 308.4 Å		1.03 Å/pix.
x 300 pix.
= 308.4 Å		1.03 Å/pix.
x 300 pix.
= 308.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.028 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.07 / Movie #1: 6.07
Minimum - Maximum-16.511019999999998 - 26.106128999999999
Average (Standard dev.)-0.22258796 (±0.9665328)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 308.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0281.0281.028
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z308.400308.400308.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-16.51126.106-0.223

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Supplemental data

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Sample components

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Entire : KvAP-6E1 Fab complex

EntireName: KvAP-6E1 Fab complex
Components
  • Complex: KvAP-6E1 Fab complex
    • Complex: KvAP
      • Protein or peptide: Voltage-gated potassium channel
    • Complex: 6E1 Fab

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Supramolecule #1: KvAP-6E1 Fab complex

SupramoleculeName: KvAP-6E1 Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 200 KDa

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Supramolecule #2: KvAP

SupramoleculeName: KvAP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all / Details: KvAP tetramer
Source (natural)Organism: Aeropyrum pernix (archaea)
Molecular weightTheoretical: 130 KDa

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Supramolecule #3: 6E1 Fab

SupramoleculeName: 6E1 Fab / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: unidentified (others)

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Macromolecule #1: Voltage-gated potassium channel

MacromoleculeName: Voltage-gated potassium channel / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Aeropyrum pernix (archaea)
Molecular weightTheoretical: 31.649207 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGGRVRNIG DVMEHPLVEL GVSYAALLSV IVVVVEYTMQ LSGEYLVRLY LVDLILVIIL WADYAYRAYK SGDPAGYVKK TLYEIPALV PAGLLALIEG HLAGLGLFRL VRLLRFLRIL LIISRGSKFL SAIADAADKI RFYHLFGAVM LTVLYGAFAI Y IVEYPDPN ...String:
MAGGRVRNIG DVMEHPLVEL GVSYAALLSV IVVVVEYTMQ LSGEYLVRLY LVDLILVIIL WADYAYRAYK SGDPAGYVKK TLYEIPALV PAGLLALIEG HLAGLGLFRL VRLLRFLRIL LIISRGSKFL SAIADAADKI RFYHLFGAVM LTVLYGAFAI Y IVEYPDPN SSIKSVFDAL WWAVVTATTV GYGDVVPATP IGKVIGIAVM LTGISALTLL IGTVSNMFQK ILVGEPEPSS SP AKLAEMV SSMSEEEFEE FVRTLKNLRR LENSMKLVPR GSRSHHHHHH

UniProtKB: Voltage-gated potassium channel

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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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMKClpotassium chloride
20.0 mMC4H11NO3Tris
0.5 mMC56H92O29Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 12 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4 seconds before plunging..
DetailsKvAP in complex with 6E1 Fab

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-50 / Number grids imaged: 2 / Number real images: 8000 / Average exposure time: 10.0 sec. / Average electron dose: 75.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1800000
Startup modelType of model: NONE
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 67000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.9.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN (ver. 9.11)
Final 3D classificationNumber classes: 3 / Avg.num./class: 32000 / Software - Name: RELION (ver. 3.0.8)

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Atomic model buiding 1

Initial model
PDB IDChain

1ors

source_name: PDB, initial_model_type: experimental model

1orq

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6uwm:
Single particle cryo-EM structure of KvAP

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