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

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Basic information

Entry
Database: PDB / ID: 6uwm
TitleSingle particle cryo-EM structure of KvAP
ComponentsVoltage-gated potassium channel
KeywordsTRANSPORT PROTEIN / voltage-gated potassium channel / non-domain-swapped
Function / homologyIon transport domain / Voltage-gated potassium channel / Ion transport protein / voltage-gated potassium channel activity / regulation of ion transmembrane transport / voltage-gated potassium channel complex / identical protein binding / Voltage-gated potassium channel
Function and homology information
Biological speciesAeropyrum pernix (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsTao, X. / MacKinnon, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM43949 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 5, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Voltage-gated potassium channel
C: Voltage-gated potassium channel
B: Voltage-gated potassium channel
D: Voltage-gated potassium channel


Theoretical massNumber of molelcules
Total (without water)126,5974
Polymers126,5974
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6840 Å2
ΔGint-77 kcal/mol
Surface area51610 Å2

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Components

#1: Protein/peptide
Voltage-gated potassium channel / / KvAP


Mass: 31649.207 Da / Num. of mol.: 4 / Fragment: UNP residues 25-295
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeropyrum pernix (archaea) / Gene: APE_0955 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9YDF8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSourceDetails
1KvAP-6E1 Fab complex10MULTIPLE SOURCES
2KvAP11RECOMBINANTKvAP tetramer
36E1 Fab1NATURAL
Molecular weight

Experimental value: NO

IDEntity assembly-IDValue (°)
110.2 MDa
220.13 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Aeropyrum pernix (archaea)56636
23unidentified (others)32644
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET28a
Buffer solutionpH: 8
Buffer component

Buffer-ID: 1

IDConc.NameFormula
1150 mMpotassium chlorideKCl
220 mMTrisC4H11NO3
30.5 mMDigitoninC56H92O29
SpecimenConc.: 6 mg/ml / Details: KvAP in complex with 6E1 Fab / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: Blot for 4 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 75 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 8000
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
1Gautomatchv0.56_sm61_cu8.0particle selection
2SerialEMimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.13.1model fitting
8Coot0.8.9.2model fitting
10PHENIX1.16model refinement
11cryoSPARC2.9.0initial Euler assignment
12FREALIGN9.11final Euler assignment
13RELION3.0.8classification
14FREALIGN9.113D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1800000
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67000 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID3D fitting-ID
11ORS1
21ORQ1

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