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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 / bioluminescence / RHOA GTPase cycle / generation of precursor metabolites and energy / ruffle / 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 / bioluminescence / RHOA GTPase cycle / generation of precursor metabolites and energy / ruffle / cellular response to mechanical stimulus / Golgi membrane / Golgi apparatus / plasma membrane / cytosol
Similarity search - Function
Transmembrane protein GPR107/GPR108-like / GOST, seven transmembrane 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: To Be Published
Title: Cryo-EM structure of human TMEM87A, PE-bound
Authors: Han, A. / Kim, H.M.
History
DepositionAug 3, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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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

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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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