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- PDB-8htt: Cryo-EM structure of human TMEM87A, gluconate-bound -

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Basic information

Entry
Database: PDB / ID: 8htt
TitleCryo-EM structure of human TMEM87A, gluconate-bound
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
D-gluconic acid / EGFP / Transmembrane protein 87A
Similarity search - Component
Biological speciesHomo sapiens (human)
Human adenovirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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
DepositionDec 21, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 27, 2023Provider: 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
B: Transmembrane protein 87A,EGFP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,5975
Polymers97,9661
Non-polymers1,6314
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transmembrane protein 87A,EGFP


Mass: 97965.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The chimera of Transmembrane protein 87A, Linkers, EGFP and Tags
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Human adenovirus 2
Gene: TMEM87A, PSEC0094 / Cell (production host): Expi293F / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q8NBN3, UniProt: A0A6M5E0N3
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Sugar ChemComp-GCO / D-gluconic acid / GLUCONIC ACID


Type: D-saccharide / Mass: 196.155 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O7 / 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 with GFP tag and Twin-strep tag with gluconate
Type: ORGANELLE OR CELLULAR COMPONENT
Details: Transmembrane protein 87A with GFP tag and Twin-strep tag with gluconate purified with detergent LMNG/CHS
Entity ID: #1 / Source: RECOMBINANT
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 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2250 mMSodium ChlorideNaCl1
30.01 % (w/v)lauryl maltose neopentyl glycolLMNG1
40.002 % (w/v)Cholesteryl hemisuccinateCHS1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: 6.14 sec. / Electron dose: 68.15 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13099
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.2particle selectionCryospatc template based auto-picking was used.
2EPU2.13image acquisition
4cryoSPARC3.3.2CTF correctionpatch CTF estimation was used.
7UCSF Chimera1.16model fittingFit in map tool
9PHENIX1.19.2model refinementReal-space refinement
10cryoSPARC3.3.2initial Euler assignmentAb-intio reconstruction
11cryoSPARC3.3.2final Euler assignmentNon-uniform refinement
12cryoSPARC3.3.2classificationHetero refinement
13cryoSPARC3.3.23D reconstructionLocal refinement
CTF correctionDetails: PatchCTF in Cryosparc was used / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6035205
Details: The previous model was used as a template for particle picking. 2D class average images were generated as templates for subsequent reference-based auto-picking.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 201915 / Algorithm: FOURIER SPACE
Details: Non-uniform refinement and CTF refinement were used to improve the particle alignment and map quality.
Num. of class averages: 1 / 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_d0.0023376
ELECTRON MICROSCOPYf_angle_d0.4754579
ELECTRON MICROSCOPYf_dihedral_angle_d10.992467
ELECTRON MICROSCOPYf_chiral_restr0.089544
ELECTRON MICROSCOPYf_plane_restr0.002537

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