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- EMDB-16311: Structure of the rod CNG channel bound to calmodulin -

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Basic information

Entry
Database: EMDB / ID: EMD-16311
TitleStructure of the rod CNG channel bound to calmodulin
Map data
Sample
  • Complex: Native CNG channel from retinal rods
    • Protein or peptide: Cyclic nucleotide-gated cation channel beta-1
    • Protein or peptide: cGMP-gated cation channel alpha-1
    • Protein or peptide: calmodulin-1
Function / homology
Function and homology information


intracellular cyclic nucleotide activated cation channel complex / intracellularly cGMP-activated cation channel activity / intracellularly cAMP-activated cation channel activity / Inactivation, recovery and regulation of the phototransduction cascade / Activation of the phototransduction cascade / response to stimulus / photoreceptor outer segment membrane / transmembrane transporter complex / monoatomic cation transmembrane transport / monoatomic cation transport ...intracellular cyclic nucleotide activated cation channel complex / intracellularly cGMP-activated cation channel activity / intracellularly cAMP-activated cation channel activity / Inactivation, recovery and regulation of the phototransduction cascade / Activation of the phototransduction cascade / response to stimulus / photoreceptor outer segment membrane / transmembrane transporter complex / monoatomic cation transmembrane transport / monoatomic cation transport / cGMP binding / photoreceptor outer segment / cAMP binding / visual perception / sensory perception of smell / membrane => GO:0016020 / protein-containing complex binding / positive regulation of gene expression / protein homodimerization activity / protein-containing complex / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Cyclic nucleotide-gated cation channel beta-1 / Cyclic nucleotide-gated channel, C-terminal leucine zipper domain / C-terminal leucine zipper domain of cyclic nucleotide-gated channels / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain ...Cyclic nucleotide-gated cation channel beta-1 / Cyclic nucleotide-gated channel, C-terminal leucine zipper domain / C-terminal leucine zipper domain of cyclic nucleotide-gated channels / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
: / cGMP-gated cation channel alpha-1 / Cyclic nucleotide-gated cation channel beta-1
Similarity search - Component
Biological speciesBos taurus (cattle) / cattle (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsMarino J
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation19082 Switzerland
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel.
Authors: Diane C A Barret / Dina Schuster / Matthew J Rodrigues / Alexander Leitner / Paola Picotti / Gebhard F X Schertler / U Benjamin Kaupp / Volodymyr M Korkhov / Jacopo Marino /
Abstract: Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation ...Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.
History
DepositionDec 8, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16311.png

Downloads & links

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Map

FileDownload / File: emd_16311.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.02 Å
Density
Contour LevelBy AUTHOR: 0.151
Minimum - Maximum-0.45458338 - 0.82537735
Average (Standard dev.)2.5526719e-05 (±0.018088501)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 326.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16311_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16311_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16311_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Native CNG channel from retinal rods

EntireName: Native CNG channel from retinal rods
Components
  • Complex: Native CNG channel from retinal rods
    • Protein or peptide: Cyclic nucleotide-gated cation channel beta-1
    • Protein or peptide: cGMP-gated cation channel alpha-1
    • Protein or peptide: calmodulin-1

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Supramolecule #1: Native CNG channel from retinal rods

SupramoleculeName: Native CNG channel from retinal rods / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 410 KDa

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Macromolecule #1: Cyclic nucleotide-gated cation channel beta-1

MacromoleculeName: Cyclic nucleotide-gated cation channel beta-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: cattle (cattle)
Molecular weightTheoretical: 155.228562 KDa
SequenceString: MLGWVQRVLP QPPGTPQKTK QEEEGTEPEP ELEPKPETAP EETELEEVSL PPEEPCVGKE VAAVTLGPQG TQETALTPPT SLQAQVSVA PEAHSSPRGW VLTWLRKGVE KVVPQPAHSS RPSQNIAAGL ESPDQQAGAQ ILGQCGTGGS DEPSEPSRAE D PGPGPWLL ...String:
MLGWVQRVLP QPPGTPQKTK QEEEGTEPEP ELEPKPETAP EETELEEVSL PPEEPCVGKE VAAVTLGPQG TQETALTPPT SLQAQVSVA PEAHSSPRGW VLTWLRKGVE KVVPQPAHSS RPSQNIAAGL ESPDQQAGAQ ILGQCGTGGS DEPSEPSRAE D PGPGPWLL RWFEQNLEKM LPQPPKISEG WRDEPTDAAL GPEPPGPALE IKPMLQAQES PSLPAPGPPE PEEEPIPEPQ PT IQASSLP PPQDSARLMA WILHRLEMAL PQPVIRGKGG EQESDAPVTC DVQTISILPG EQEESHLILE EVDPHWEEDE HQE GSTSTS PRTSEAAPAD EEKGKVVEQT PRELPRIQEE KEDEEEEKED GEEEEEEGRE KEEEEGEEKE EEEGREKEEE EGEK KEEEG REKEEEEGGE KEDEEGREKE EEEGRGKEEE EGGEKEEEEG RGKEEVEGRE EEEDEEEEQD HSVLLDSYLV PQSEE DRSE ESETQDQSEV GGAQAQGEVG GAQALSEESE TQDQSEVGGA QDQSEVGGAQ AQGEVGGAQE QDGVGGAQDQ STSHQE LQE EALADSSGVP ATEEHPELQV EDADADSRPL IAEENPPSPV QLPLSPAKSD TLAVPGSATG SLRKRLPSQD DEAEELK ML SPAASPVVAW SDPTSPQGTD DQDRATSTAS QNSAIINDRL QELVKLFKER TEKVKEKLID PDVTSDEESP KPSPAKKA P EPAPEVKPAE AGQVEEEHYC EMLCCKFKRR PWKKYQFPQS IDPLTNLMYI LWLFFVVLAW NWNCWLIPVR WAFPYQTPD NIHLWLLMDY LCDLIYLLDI TVFQMRLQFV RGGDIITDKK EMRNNYVKSQ RFKMDMLCLL PLDLLYLKFG VNPLLRLPRC LKYMAFFEF NNRLESILSK AYVYRVIRTT AYLLYSLHLN SCLYYWASAY EGLGSTHWVY DGVGNSYIRC YYWAVKTLIT I GGLPDPRT LFEIVFQGLN YFTGVFAFSV MIGQMRDVVG AATAGQTYYR SCMDSTVKYM NFYKIPRSVQ NRVKTWYEYT WH SQGMLDE SELMVQLPDK MRLDLAIDVN YSIVSKVALF QGCDRQMIFD MLKRLRSVVY LPNDYVCKKG EIGREMYIIQ AGQ VQVLGG PDGKSVLVTL KAGSVFGEIS LLAVGGGNRR TANVVAHGFT NLFILDKKDL NEILVHYPES QKLLRKKARR MLRN NNKPK EKSVLILPPR AGTPKLFNAA LAAAGKMGAK GGRGGRLALL RARLKELAAL EAAARQQQLL EQAKSSEDAA VGEEG SASP EQPPRPEPPA PEAPAPEPTA PEPLAPEAPA PEAPAPSSPP PASQERPEGD KDAARPEEHP VRIHVTLGPD PSEQIL LVE VPEKQEEKEK KEEETEEKEE GEEARKEKEE E

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Macromolecule #2: cGMP-gated cation channel alpha-1

MacromoleculeName: cGMP-gated cation channel alpha-1 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: cattle (cattle)
Molecular weightTheoretical: 79.712164 KDa
SequenceString: MKKVIINTWH SFVNIPNVVG PDVEKEITRM ENGACSSFSG DDDDSASMFE ESETENPHAR DSFRSNTHGS GQPSQREQYL PGAIALFNV NNSSNKEQEP KEKKKKKKEK KSKPDDKNEN KKDPEKKKKK EKDKDKKKKE EKGKDKKEEE KKEVVVIDPS G NTYYNWLF ...String:
MKKVIINTWH SFVNIPNVVG PDVEKEITRM ENGACSSFSG DDDDSASMFE ESETENPHAR DSFRSNTHGS GQPSQREQYL PGAIALFNV NNSSNKEQEP KEKKKKKKEK KSKPDDKNEN KKDPEKKKKK EKDKDKKKKE EKGKDKKEEE KKEVVVIDPS G NTYYNWLF CITLPVMYNW TMIIARACFD ELQSDYLEYW LAFDYLSDVV YLLDMFVRTR TGYLEQGLLV KEERKLIDKY KS TFQFKLD VLSVIPTDLL YIKFGWNYPE IRLNRLLRIS RMFEFFQRTE TRTNYPNIFR ISNLVMYIII IIHWNACVYF SIS KAIGFG NDTWVYPDVN DPDFGRLARK YVYSLYWSTL TLTTIGETPP PVRDSEYFFV VADFLIGVLI FATIVGNIGS MISN MNAAR AEFQARIDAI KQYMHFRNVS KDMEKRVIKW FDYLWTNKKT VDEREVLKYL PDKLRAEIAI NVHLDTLKKV RIFAD CEAG LLVELVLKLQ PQVYSPGDYI CKKGDIGREM YIIKEGKLAV VADDGITQFV VLSDGSYFGE ISILNIKGSK AGNRRT ANI KSIGYSDLFC LSKDDLMEAL TEYPDAKGML EEKGKQILMK DGLLDINIAN AGSDPKDLEE KVTRMESSVD LLQTRFA RI LAEYESMQQK LKQRLTKVEK FLKPLIDTEF SAIEGSGTES GPTDSTQD

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Macromolecule #3: calmodulin-1

MacromoleculeName: calmodulin-1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: cattle (cattle)
Molecular weightTheoretical: 16.852545 KDa
SequenceString:
MADQLTEEQI AEFKEAFSLF DKDGDGTITT KELGTVMRSL GQNPTEAELQ DMINEVDADG NGTIDFPEFL TMMARKMKDT DSEEEIREA FRVFDKDGNG YISAAELRHV MTNLGEKLTD EEVDEMIREA DIDGDGQVNY EEFVQMMTAK

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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

Concentration0.3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 2 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 62.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 279337
FSC plot (resolution estimation)

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