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- EMDB-23746: Cryo-EM structure of zebrafish TRPM5 E337A mutant in the presence... -

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Basic information

Entry
Database: EMDB / ID: EMD-23746
TitleCryo-EM structure of zebrafish TRPM5 E337A mutant in the presence of 5 mM calcium (low calcium occupancy in the transmembrane domain)
Map dataTetrameric map of zebrafish TRPM5 E337A mutant with low calcium occupancy in the transmembrane domain. The map is only used for docking a tetramer model. The associated subunit map is also provided.
Sample
  • Complex: TRPM5 channel E337A mutant
    • Protein or peptide: Transient receptor potential melastatin 5
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2R)-2-(hydroxymethyl)-4-{[(25R)-10alpha,14beta,17beta-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside
  • Ligand: (25R)-14beta,17beta-spirost-5-en-3beta-ol
Function / homology
Function and homology information


ligand-gated calcium channel activity / calcium-activated cation channel activity / calcium ion transmembrane transport / membrane
Similarity search - Function
Transient receptor potential channel, canonical / TRPM, SLOG domain / SLOG in TRPM / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Transient receptor potential melastatin 5
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM
AuthorsRuan Z / Lu W / Du J / Haley E
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R56HL144929 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS111031 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL153219 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS112363 United States
American Heart Association20POST35120556 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structures of the TRPM5 channel elucidate mechanisms of activation and inhibition.
Authors: Zheng Ruan / Emery Haley / Ian J Orozco / Mark Sabat / Richard Myers / Rebecca Roth / Juan Du / Wei Lü /
Abstract: The Ca-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca regulates TRPM5 activity remains elusive. We report cryo-EM ...The Ca-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca site (Ca) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The Ca site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca binding to the Ca site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.
History
DepositionApr 1, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7mbt
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23746.png

Downloads & links

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Map

FileDownload / File: emd_23746.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTetrameric map of zebrafish TRPM5 E337A mutant with low calcium occupancy in the transmembrane domain. The map is only used for docking a tetramer model. The associated subunit map is also provided.
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.013
Minimum - Maximum-0.019452771 - 0.039720297
Average (Standard dev.)0.00029161858 (±0.0025911725)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8260.8260.826
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z297.360297.360297.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-210-210-210
NX/NY/NZ420420420
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0190.0400.000

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Supplemental data

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Additional map: Sharpened single subunit map for zebrafish TRPM5 E337A...

Fileemd_23746_additional_1.map
AnnotationSharpened single subunit map for zebrafish TRPM5 E337A mutant with low calcium occupancy at transmembrane domain.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non-sharpened single subunit map for zebrafish TRPM5 E337A...

Fileemd_23746_additional_2.map
AnnotationNon-sharpened single subunit map for zebrafish TRPM5 E337A mutant with low calcium occupancy at transmembrane domain.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TRPM5 channel E337A mutant

EntireName: TRPM5 channel E337A mutant
Components
  • Complex: TRPM5 channel E337A mutant
    • Protein or peptide: Transient receptor potential melastatin 5
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2R)-2-(hydroxymethyl)-4-{[(25R)-10alpha,14beta,17beta-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside
  • Ligand: (25R)-14beta,17beta-spirost-5-en-3beta-ol

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Supramolecule #1: TRPM5 channel E337A mutant

SupramoleculeName: TRPM5 channel E337A mutant / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Transient receptor potential melastatin 5

MacromoleculeName: Transient receptor potential melastatin 5 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 132.740953 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVEKSSERFD KQMAGRLGDI DFTGVSRTRG KFVRVTSSTD PAEIYQILTK QWGLAPPHLV VALMGGDEVA QLKPWLRDTL RKGLVKAAQ STGAWILTSG LRFGITKNLG QAVRDHSLAS TSPKVRVVAI GIAPWNMIQN RDLLLSAKPD HPATYPTEDL P YGAVYSLD ...String:
MVEKSSERFD KQMAGRLGDI DFTGVSRTRG KFVRVTSSTD PAEIYQILTK QWGLAPPHLV VALMGGDEVA QLKPWLRDTL RKGLVKAAQ STGAWILTSG LRFGITKNLG QAVRDHSLAS TSPKVRVVAI GIAPWNMIQN RDLLLSAKPD HPATYPTEDL P YGAVYSLD CNHSHFILVD EDPKRPGATG EMRVKMLKHI SLQRTGYGGT GSIEIPVLCL LVHGEPRILQ KMYKNIQNSI PW LILAGSG GVADILVTLM DRGCWDADIV QELLINTFPD GLHSTEITSW TKLIQRILDH GHLLTVHDPE QDSELDTVIL KAL VKACKS QSQEAQDFLD ALKLAVAWNR VDIAKSEIFS GDVQWSAQDL EEVMMEALVN DKPDFVRLFV DNGVNIKQFL TYGR LQELY CSVSEKNLLH TLLLKKNQER QAQLARKRMS GNPNNELGDR KFRFTFHEVS KVLKDFLDDT CKGFYQKLPA ERMGK GRLF HSQKNLPDMD RRCEHPWRDL FLWAILQNRQ EMANYFWAMG PEAVAAALVG CKIMKEMAHL ATEAESARSM KNAKYE QFA MDLFSECYSN SEDRAYSLLV RKTCCWSKAT VLNIATLAEA KCFFAHDGVQ ALLTKVWWGA MRTDTSISRL VLTFFIP PL VWTSLIKFNP EEQVSKDEGE PFAELDSLET EQALLLTDGD PVAGEGSAET AARSCSATFI RVVLRRWNRF WSAPVTVF M GNVIMYFAFL ILFSYVLLLD FRPPPPYGPS AAEIILYFWV FTLVLEEIRQ SFFTDEDMSI LKKMKLYVED NWNKCDMVA ISLFVVGLSC RMAMSTYEAG RTVLALDFMV FTLRLIHIFA IHKQLGPKII IVERMIKDVF FFLFFLSVWL IAYGVTTQAL LHPNDPRID WVFRRALYRP YLHIFGQIPL EEIDAAKMPD DNCTTDVQEI ILGTLPPCPN IYANWLVILL LVIYLLVTNV L LLNLLIAM FSYTFQVVQE NADIFWKFQR YNLIVEYHSR PALAPPFIII SHITQALLSF IKKTENTQDL LERELPSGLD QK LMTWETV QKENYLAKLE HEHRESSGER LRYTSSKVQT LLRMVGGFKD QEKRMATVET EVRYCGEVLS WIAECFHKST LKC DRDAPK APRSIAGSSR DQQPQGAKRQ QPGGHPAYGT DKKLPFIDH

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #3: (2R)-2-(hydroxymethyl)-4-{[(25R)-10alpha,14beta,17beta-spirost-5-...

MacromoleculeName: (2R)-2-(hydroxymethyl)-4-{[(25R)-10alpha,14beta,17beta-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside
type: ligand / ID: 3 / Number of copies: 4 / Formula: YUY
Molecular weightTheoretical: 841.033 Da
Chemical component information

ChemComp-YUY:
(2R)-2-(hydroxymethyl)-4-{[(25R)-10alpha,14beta,17beta-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside

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Macromolecule #4: (25R)-14beta,17beta-spirost-5-en-3beta-ol

MacromoleculeName: (25R)-14beta,17beta-spirost-5-en-3beta-ol / type: ligand / ID: 4 / Number of copies: 4 / Formula: YUV
Molecular weightTheoretical: 414.621 Da
Chemical component information

ChemComp-YUV:
(25R)-14beta,17beta-spirost-5-en-3beta-ol / Diosgenin

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber images used: 208000

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7mbt:
Cryo-EM structure of zebrafish TRPM5 E337A mutant in the presence of 5 mM calcium (low calcium occupancy in the transmembrane domain)

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