[English] 日本語
Yorodumi
- PDB-8qz4: Crystal structure of human two pore domain potassium ion channel ... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8qz4
TitleCrystal structure of human two pore domain potassium ion channel TREK-2 (K2P10.1) in complex with an activatory nanobody (Nb76)
Components
  • Nanobody 76
  • Potassium channel subfamily K member 10
KeywordsMEMBRANE PROTEIN / Potassium ion channel / Nanobody / Structural Genomics / Structural Genomics Consortium / SGC
Function / homology
Function and homology information


TWIK related potassium channel (TREK) / Phase 4 - resting membrane potential / stabilization of membrane potential / potassium ion leak channel activity / outward rectifier potassium channel activity / monoatomic ion channel complex / potassium channel activity / potassium ion transmembrane transport / memory / signal transduction / plasma membrane
Similarity search - Function
Two pore domain potassium channel, TREK / Two pore domain potassium channel / Potassium channel domain / Ion channel
Similarity search - Domain/homology
: / CHOLESTEROL HEMISUCCINATE / Potassium channel subfamily K member 10
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsRodstrom, K.E.J. / Pike, A.C.W. / Baronina, A. / Ang, J. / Bushell, S.R. / Chalk, R. / Mukhopadhyay, S.M.M. / Pardon, E. / Arrowsmith, C.H. / Edwards, A.M. ...Rodstrom, K.E.J. / Pike, A.C.W. / Baronina, A. / Ang, J. / Bushell, S.R. / Chalk, R. / Mukhopadhyay, S.M.M. / Pardon, E. / Arrowsmith, C.H. / Edwards, A.M. / Bountra, C. / Burgess-Brown, N.A. / Tucker, S.J. / Steyaert, J. / Carpenter, E.P. / Structural Genomics Consortium (SGC)
Funding support United Kingdom, France, Belgium, 8items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T002018/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S008608/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/W017741/1 United Kingdom
Grenoble Instruct-ERIC Center (ISBG) France
Research Foundation - Flanders (FWO) Belgium
Wellcome Trust106169/Z/14/Z United Kingdom
Wellcome TrustWT084655MA United Kingdom
Wellcome Trust102161/B/13/Z United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation.
Authors: Karin E J Rödström / Alexander Cloake / Janina Sörmann / Agnese Baronina / Kathryn H M Smith / Ashley C W Pike / Jackie Ang / Peter Proks / Marcus Schewe / Ingelise Holland-Kaye / Simon R ...Authors: Karin E J Rödström / Alexander Cloake / Janina Sörmann / Agnese Baronina / Kathryn H M Smith / Ashley C W Pike / Jackie Ang / Peter Proks / Marcus Schewe / Ingelise Holland-Kaye / Simon R Bushell / Jenna Elliott / Els Pardon / Thomas Baukrowitz / Raymond J Owens / Simon Newstead / Jan Steyaert / Elisabeth P Carpenter / Stephen J Tucker /
Abstract: Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic ...Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
History
DepositionOct 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Potassium channel subfamily K member 10
B: Potassium channel subfamily K member 10
C: Nanobody 76
D: Nanobody 76
hetero molecules


Theoretical massNumber of molelcules
Total (without water)91,91312
Polymers89,4174
Non-polymers2,4968
Water1448
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15270 Å2
ΔGint-155 kcal/mol
Surface area32120 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.28, 75.576, 259.15
Angle α, β, γ (deg.)90, 90, 90
Int Tables number19
Space group name H-MP212121

-
Components

#1: Protein Potassium channel subfamily K member 10 / Outward rectifying potassium channel protein TREK-2 / TREK-2 K(+) channel subunit


Mass: 31060.111 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNK10, TREK2 / Plasmid: pFB-CT10HF-LIC / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P57789
#2: Antibody Nanobody 76


Mass: 13648.158 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli) / Strain (production host): WK6
#3: Chemical
ChemComp-BA / BARIUM ION


Mass: 137.327 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ba
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

-
Experimental details

-
Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

-
Sample preparation

CrystalDensity Matthews: 3.74 Å3/Da / Density % sol: 67.17 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 0.1 M HEPES pH 7.0, 0.3 M BaCl2, 39% polyethylene glycol 400

-
Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9687 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jul 2, 2017
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9687 Å / Relative weight: 1
ReflectionResolution: 3.203→51.83 Å / Num. obs: 17904 / % possible obs: 77.9 % / Redundancy: 4 % / CC1/2: 0.998 / Rpim(I) all: 0.066 / Rrim(I) all: 0.134 / Net I/σ(I): 8.4
Reflection shellResolution: 3.203→3.336 Å / Redundancy: 4 % / Mean I/σ(I) obs: 1.7 / Num. unique obs: 892 / CC1/2: 0.615 / Rpim(I) all: 0.522 / Rrim(I) all: 1.065 / % possible all: 34.4

-
Processing

Software
NameVersionClassification
BUSTER2.10.4refinement
XDSdata reduction
Aimlessdata scaling
STARANISOdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.2→31.11 Å / Cor.coef. Fo:Fc: 0.849 / Cor.coef. Fo:Fc free: 0.804 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.562
RfactorNum. reflection% reflectionSelection details
Rfree0.2984 894 -RANDOM
Rwork0.2704 ---
obs0.2718 17829 77.8 %-
Displacement parametersBiso mean: 77.01 Å2
Baniso -1Baniso -2Baniso -3
1--2.5586 Å20 Å20 Å2
2---4.3165 Å20 Å2
3---6.8751 Å2
Refine analyzeLuzzati coordinate error obs: 0.56 Å
Refinement stepCycle: LAST / Resolution: 3.2→31.11 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5321 0 144 8 5473
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0045602HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.657693HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1767SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes929HARMONIC5
X-RAY DIFFRACTIONt_it5602HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion789SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact4390SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion1.98
X-RAY DIFFRACTIONt_other_torsion15.71
LS refinement shellResolution: 3.2→3.26 Å
RfactorNum. reflection% reflection
Rfree0.4292 24 -
Rwork0.3525 --
obs0.3576 406 30.89 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.01130.8916-0.69932.0778-1.53065.58910.009-0.17430.6076-0.17430.1329-0.92030.6076-0.9203-0.1418-0.14050.1724-0.0339-0.0450.09740.162115.03942.1855292.451
21.03360.73030.32863.6154-2.44914.9825-0.01520.15960.11170.1596-0.3489-0.01140.1117-0.01140.3641-0.28660.1514-0.016-0.22150.13120.060423.1295.7657302.593
311.0516-2.27061.87424.9655-1.409113.9225-0.49750.00240.11280.00240.3567-0.3650.1128-0.3650.1408-0.4506-0.11140.0427-0.336-0.0464-0.388713.688419.5136263.577
46.4370.26420.24647.48211.0758.3136-0.76720.0999-0.71510.09990.38871.6327-0.71511.63270.3784-0.01850.2547-0.32760.2756-0.0623-0.018747.033828.2094287.131
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|71 - A|337 A|401 - A|402 B|401 - B|401 }A71 - 337
2X-RAY DIFFRACTION1{ A|71 - A|337 A|401 - A|402 B|401 - B|401 }A401 - 402
3X-RAY DIFFRACTION1{ A|71 - A|337 A|401 - A|402 B|401 - B|401 }B401
4X-RAY DIFFRACTION2{ B|74 - B|337 }B74 - 337
5X-RAY DIFFRACTION3{ C|1 - C|112 }C1 - 112
6X-RAY DIFFRACTION4{ D|1 - D|112 }D1 - 112

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more