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- EMDB-37069: Cryo-EM structure of human TMEM87A A308M -

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Basic information

Entry
Database: EMDB / ID: EMD-37069
TitleCryo-EM structure of human TMEM87A A308M
Map datacryo-EM map of human TMEM87A with A308M mutation
Sample
  • Organelle or cellular component: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag
    • Protein or peptide: Transmembrane protein 87A,EGFP
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (9R,12R)-15-amino-12-hydroxy-6,12-dioxo-7,11,13-trioxa-12lambda~5~-phosphapentadecan-9-yl undecanoate
Keywordsnon-selective cation channel / ion channel / membrane protein / Golgi-localized protein
Function / homology
Function and homology information


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 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
EGFP / Transmembrane protein 87A
Similarity search - Component
Biological speciesHomo sapiens (human) / Human adenovirus C serotype 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsHan A / Kim HM
Funding support Korea, Republic Of, 1 items
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 HM
History
DepositionAug 3, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBj / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_37069.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM map of human TMEM87A with A308M mutation
Voxel sizeX=Y=Z: 0.848 Å
Density
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.34204993 - 0.6448246
Average (Standard dev.)-0.000028712942 (±0.012783905)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 237.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Transmembrane protein 87A A308M with GFP tag and Twin-strep tag

EntireName: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag
Components
  • Organelle or cellular component: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag
    • Protein or peptide: Transmembrane protein 87A,EGFP
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (9R,12R)-15-amino-12-hydroxy-6,12-dioxo-7,11,13-trioxa-12lambda~5~-phosphapentadecan-9-yl undecanoate

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Supramolecule #1: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag

SupramoleculeName: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Transmembrane protein 87A A308M with GFP tag and Twin-strep tag purified with detergent LMNG/CHS
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Transmembrane protein 87A,EGFP

MacromoleculeName: Transmembrane protein 87A,EGFP / type: protein_or_peptide / ID: 1
Details: Transmembrane protein 87A A308M with GFP and twin strep tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human adenovirus C serotype 2
Molecular weightTheoretical: 96.983539 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAAAAWLQVL PVILLLLGAH PSPLSFFSAG PATVAAADRS KWHIPIPSGK NYFSFGKILF RNTTIFLKFD GEPCDLSLNI TWYLKSADC YNEIYNFKAE EVELYLEKLK EKRGLSGKYQ TSSKLFQNCS ELFKTQTFSG DFMHRLPLLG EKQEAKENGT N LTFIGDKT ...String:
MAAAAWLQVL PVILLLLGAH PSPLSFFSAG PATVAAADRS KWHIPIPSGK NYFSFGKILF RNTTIFLKFD GEPCDLSLNI TWYLKSADC YNEIYNFKAE EVELYLEKLK EKRGLSGKYQ TSSKLFQNCS ELFKTQTFSG DFMHRLPLLG EKQEAKENGT N LTFIGDKT AMHEPLQTWQ DAPYIFIVHI GISSSKESSK ENSLSNLFTM TVEVKGPYEY LTLEDYPLMI FFMVMCIVYV LF GVLWLAW SACYWRDLLR IQFWIGAVIF LGMLEKAVFY AEFQNIRYKG ESVQGALILA ELLSAVKRSL MRTLVIIVSL GYG IVKPRL GVTLHKVVVA GALYLLFSGM EGVLRVTGAQ TDLASLAFIP LAFLDTALCW WIFISLTQTM KLLKLRRNIV KLSL YRHFT NTLILAVAAS IVFIIWTTMK FRIVTCQSDW RELWVDDAIW RLLFSMILFV IMVLWRPSAN NQRFAFSPLS EEEEE DEQK EPMLKESFEG MKMRSTKQEP NGNSKVNKAQ EDDLKWVEEN VPSSVTDVAL PALLDSDEER MITHFERSKM ELKENL YFQ GNPAFLYKVV DPVVSKGEEL FTGVVPILVE LDGDVNGHKF SVSGEGEGDA TYGKLTLKFI CTTGKLPVPW PTLVTTL TY GVQCFSRYPD HMKQHDFFKS AMPEGYVQER TIFFKDDGNY KTRAEVKFEG DTLVNRIELK GIDFKEDGNI LGHKLEYN Y NSHNVYIMAD KQKNGIKVNF KIRHNIEDGS VQLADHYQQN TPIGDGPVLL PDNHYLSTQS ALSKDPNEKR DHMVLLEFV TAAGITLGMD ELYKEFLVPR GSSRSAWSHP QFEKGGGSGG GSGGSAWSHP QFEK

UniProtKB: Transmembrane protein 87A, EGFP

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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: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: (9R,12R)-15-amino-12-hydroxy-6,12-dioxo-7,11,13-trioxa-12lambda~5...

MacromoleculeName: (9R,12R)-15-amino-12-hydroxy-6,12-dioxo-7,11,13-trioxa-12lambda~5~-phosphapentadecan-9-yl undecanoate
type: ligand / ID: 3 / Number of copies: 1 / Formula: 65I
Molecular weightTheoretical: 481.56 Da
Chemical component information

ChemComp-65I:
(9R,12R)-15-amino-12-hydroxy-6,12-dioxo-7,11,13-trioxa-12lambda~5~-phosphapentadecan-9-yl undecanoate

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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

Concentration0.8 mg/mL
BufferpH: 9
Component:
ConcentrationFormulaName
50.0 mMHEPEStrisaminomethane
250.0 mMNaClSodium Chloride
0.01 % (w/v)DDMDodecylmaltoside
0.002 % (w/v)CHSCholesteryl hemisuccinate

Details: 50mM HEPES pH 7.5, 250mM NaCl, 0.01% (w/v) LMNG, 0.002% (w/v) CHS
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.015 kPa
Details: The grid was negatively glow-discharged for 60 sec with 15mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureComa free - Residual tilt: 10.0 mrad
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsUsing Zemlin tableau in sherpa
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 4565 / Average exposure time: 6.09 sec. / Average electron dose: 67.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 58900 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8hsi

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Local refinement / Number images used: 360876
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Ab-intio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Non-uniform refinement
Final 3D classificationNumber classes: 6 / Avg.num./class: 793056 / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Hetero refinement

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Atomic model buiding 1

DetailsChimera 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 40
Output model

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

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