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- PDB-8hsi: Cryo-EM structure of human TMEM87A, PE-bound -

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Basic information

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


retrograde transport, endosome to Golgi / Golgi cisterna membrane / RHOA GTPase cycle / 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
Similarity search - Domain/homology
CHOLESTEROL / Chem-L9Q / Transmembrane protein 87A
Similarity search - Component
Biological speciesHomo sapiens (human)
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
DepositionDec 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transmembrane protein 87A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,4497
Polymers63,4951
Non-polymers2,9556
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


Mass: 63494.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM87A, PSEC0094 / Cell (production host): Expi293F / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q8NBN3
#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: Chemical ChemComp-L9Q / (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine


Mass: 746.050 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H80NO8P / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O / 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 with GFP tag and Twin-strep tag
Type: ORGANELLE OR CELLULAR COMPONENT
Details: Transmembrane protein 87A with GFP tag and Twin-strep tag 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 Tris pH 9.0, 250mM NaCl, 0.01% (w/v) LMNG, 0.002% (w/v) CHS
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtrisaminomethaneTris1
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: 15mA / Grid 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: 67.72 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10377
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_4092: / 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
9cryoSPARC3.3.2initial Euler assignmentAb-intio reconstruction
10cryoSPARC3.3.2final Euler assignmentNon-uniform refinement
11cryoSPARC3.3.2classificationHetero refinement
12cryoSPARC3.3.23D reconstructionLocal refinement
13PHENIX1.19.2model refinementReal-space 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.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 445198 / 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_d0.0043619
ELECTRON MICROSCOPYf_angle_d1.0814908
ELECTRON MICROSCOPYf_dihedral_angle_d16.234539
ELECTRON MICROSCOPYf_chiral_restr0.285577
ELECTRON MICROSCOPYf_plane_restr0.004567

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