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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 / homologymonoatomic ion channel complex / potassium channel activity / Ion transport domain / Ion transport protein / identical protein binding / plasma membrane / 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 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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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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


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6840 Å2
ΔGint-77 kcal/mol
Surface area51610 Å2

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Components

#1: Protein
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
IDNameTypeEntity IDParent-IDSourceDetails
1KvAP-6E1 Fab complexCOMPLEXall0MULTIPLE SOURCES
2KvAPCOMPLEXall1RECOMBINANTKvAP tetramer
36E1 FabCOMPLEX1NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.2 MDaNO
220.13 MDaNO
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
IDConc.NameFormulaBuffer-ID
1150 mMpotassium chlorideKCl1
220 mMTrisC4H11NO31
30.5 mMDigitoninC56H92O291
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: KvAP in complex with 6E1 Fab
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 / Specimen holder 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-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11ORS

1ors

11ORS1PDBexperimental model
21ORQ

1orq

11ORQ2PDBexperimental model

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