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- PDB-8kb4: Cryo-EM structure of human TMEM87A A308M -

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Basic information

Entry
Database: PDB / ID: 8kb4
TitleCryo-EM structure of human TMEM87A A308M
ComponentsTransmembrane protein 87A,EGFP
KeywordsMEMBRANE PROTEIN / non-selective cation channel / ion channel / Golgi-localized protein
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception of touch / retrograde transport, endosome to Golgi / Golgi cisterna membrane / RHOA GTPase cycle / ruffle / bioluminescence / generation of precursor metabolites and energy / cellular response to mechanical stimulus / Golgi membrane / Golgi apparatus ...detection of mechanical stimulus involved in sensory perception of touch / retrograde transport, endosome to Golgi / Golgi cisterna membrane / RHOA GTPase cycle / ruffle / bioluminescence / generation of precursor metabolites and energy / cellular response to mechanical stimulus / Golgi membrane / Golgi apparatus / plasma membrane / cytosol
Similarity search - Function
Transmembrane protein GPR107/GPR108-like / : / : / GOST, seven transmembrane domain / TMEM87A/B, GOLD domain / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Chem-65I / EGFP / Transmembrane protein 87A
Similarity search - Component
Biological speciesHomo sapiens (human)
Human adenovirus C serotype 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsHan, A. / Kim, H.M.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
Other governmentIBS-R030-C1 Korea, Republic Of
CitationJournal: Nat Commun / Year: 2024
Title: GolpHCat (TMEM87A), a unique voltage-dependent cation channel in Golgi apparatus, contributes to Golgi-pH maintenance and hippocampus-dependent memory.
Authors: Hyunji Kang / Ah-Reum Han / Aihua Zhang / Heejin Jeong / Wuhyun Koh / Jung Moo Lee / Hayeon Lee / Hee Young Jo / Miguel A Maria-Solano / Mridula Bhalla / Jea Kwon / Woo Suk Roh / Jimin Yang ...Authors: Hyunji Kang / Ah-Reum Han / Aihua Zhang / Heejin Jeong / Wuhyun Koh / Jung Moo Lee / Hayeon Lee / Hee Young Jo / Miguel A Maria-Solano / Mridula Bhalla / Jea Kwon / Woo Suk Roh / Jimin Yang / Hyun Joo An / Sun Choi / Ho Min Kim / C Justin Lee /
Abstract: Impaired ion channels regulating Golgi pH lead to structural alterations in the Golgi apparatus, such as fragmentation, which is found, along with cognitive impairment, in Alzheimer's disease. ...Impaired ion channels regulating Golgi pH lead to structural alterations in the Golgi apparatus, such as fragmentation, which is found, along with cognitive impairment, in Alzheimer's disease. However, the causal relationship between altered Golgi structure and cognitive impairment remains elusive due to the lack of understanding of ion channels in the Golgi apparatus of brain cells. Here, we identify that a transmembrane protein TMEM87A, renamed Golgi-pH-regulating cation channel (GolpHCat), expressed in astrocytes and neurons that contributes to hippocampus-dependent memory. We find that GolpHCat displays unique voltage-dependent currents, which is potently inhibited by gluconate. Additionally, we gain structural insights into the ion conduction through GolpHCat at the molecular level by determining three high-resolution cryogenic-electron microscopy structures of human GolpHCat. GolpHCat-knockout mice show fragmented Golgi morphology and altered protein glycosylation and functions in the hippocampus, leading to impaired spatial memory. These findings suggest a molecular target for Golgi-related diseases and cognitive impairment.
History
DepositionAug 3, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transmembrane protein 87A,EGFP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,1295
Polymers96,9841
Non-polymers1,1454
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transmembrane protein 87A,EGFP


Mass: 96983.539 Da / Num. of mol.: 1 / Mutation: A308M
Source method: isolated from a genetically manipulated source
Details: Transmembrane protein 87A A308M with GFP and twin strep tag
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human adenovirus C serotype 2
Gene: TMEM87A / Cell (production host): Expi293F / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q8NBN3, UniProt: A0A6M5E0N3
#2: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-65I / (9R,12R)-15-amino-12-hydroxy-6,12-dioxo-7,11,13-trioxa-12lambda~5~-phosphapentadecan-9-yl undecanoate


Mass: 481.560 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C22H44NO8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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

ComponentName: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag
Type: ORGANELLE OR CELLULAR COMPONENT
Details: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag purified with detergent LMNG/CHS
Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F / Plasmid: pcDNA3.4 TOPO
Buffer solutionpH: 9
Details: 50mM HEPES pH 7.5, 250mM NaCl, 0.01% (w/v) LMNG, 0.002% (w/v) CHS
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtrisaminomethaneHEPES1
2250 mMSodium ChlorideNaCl1
30.01 % (w/v)DodecylmaltosideDDM1
40.002 % (w/v)Cholesteryl hemisuccinateCHS1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: The grid was negatively glow-discharged for 60 sec with 15mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Using Zemlin tableau in sherpa
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 58900 X / Nominal defocus max: 1900 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 / Residual tilt: 10 mradians
Image recordingAverage exposure time: 6.09 sec. / Electron dose: 67.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4565
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionCryospatc template based auto-picking was used.
2EPU2.13image acquisition
4cryoSPARC4.2.1CTF correctionpatch CTF estimation was used.
7UCSF Chimera1.16model fittingFit in map tool
9PHENIX1.19.2model refinementReal-space refinement
10cryoSPARC4.2.1initial Euler assignmentAb-intio reconstruction
11cryoSPARC4.2.1final Euler assignmentNon-uniform refinement
12cryoSPARC4.2.1classificationHetero refinement
13cryoSPARC4.2.13D reconstructionLocal refinement
CTF correctionDetails: PatchCTF in Cryosparc was used / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8101104
Details: 96,330 particles were first picked using a blob picker of cryoSPARC. 2D class average images were generated as templates for subsequent reference-based auto-picking.
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 360876 / Symmetry type: POINT
Atomic model buildingB value: 40 / Protocol: RIGID BODY FIT / Space: REAL
Details: Chimera Fit in map tool was used for initial local fitting. Then, Real-space refinement with the rigid body option in PHENIX was used for flexible fitting.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0043454
ELECTRON MICROSCOPYf_angle_d0.4434677
ELECTRON MICROSCOPYf_dihedral_angle_d11.87471
ELECTRON MICROSCOPYf_chiral_restr0.036535
ELECTRON MICROSCOPYf_plane_restr0.003555

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