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- PDB-5kc1: Structure of the C-terminal dimerization domain of Atg38 -

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Basic information

Entry
Database: PDB / ID: 5kc1
TitleStructure of the C-terminal dimerization domain of Atg38
ComponentsAutophagy-related protein 38
KeywordsENDOCYTOSIS / Atg38 / coiled-coil / dimerization / NRBF2 / autophagy
Function / homology
Function and homology information


phosphatidylinositol 3-kinase complex, class III, type I / phagophore assembly site membrane / phagophore assembly site / vacuolar membrane / macroautophagy / structural molecule activity / identical protein binding / cytoplasm
Similarity search - Function
IODIDE ION / AMMONIUM ION / NITRATE ION / Autophagy-related protein 38
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.2 Å
AuthorsOhashi, Y. / Soler, N. / Garcia-Ortegon, M. / Zhang, L. / Perisic, O. / Masson, G.R. / Johnson, C.M. / Williams, R.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184308 United Kingdom
CitationJournal: Autophagy / Year: 2016
Title: Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex.
Authors: Yohei Ohashi / Nicolas Soler / Miguel García Ortegón / Lufei Zhang / Marie L Kirsten / Olga Perisic / Glenn R Masson / John E Burke / Arjen J Jakobi / Apostolos A Apostolakis / Christopher ...Authors: Yohei Ohashi / Nicolas Soler / Miguel García Ortegón / Lufei Zhang / Marie L Kirsten / Olga Perisic / Glenn R Masson / John E Burke / Arjen J Jakobi / Apostolos A Apostolakis / Christopher M Johnson / Maki Ohashi / Nicholas T Ktistakis / Carsten Sachse / Roger L Williams /
Abstract: The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization ...The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.
History
DepositionJun 4, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Oct 5, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2016Group: Database references
Revision 1.2Nov 9, 2016Group: Database references
Revision 2.0Sep 13, 2017Group: Atomic model / Author supporting evidence / Derived calculations
Category: atom_site / pdbx_audit_support ...atom_site / pdbx_audit_support / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _pdbx_audit_support.funding_organization / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_site.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_seq_id / _struct_site_gen.auth_asym_id / _struct_site_gen.auth_seq_id / _struct_site_gen.label_asym_id
Revision 2.1May 8, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr1_symmetry / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Autophagy-related protein 38
D: Autophagy-related protein 38
A: Autophagy-related protein 38
B: Autophagy-related protein 38
G: Autophagy-related protein 38
H: Autophagy-related protein 38
K: Autophagy-related protein 38
L: Autophagy-related protein 38
E: Autophagy-related protein 38
F: Autophagy-related protein 38
I: Autophagy-related protein 38
J: Autophagy-related protein 38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)314,56963
Polymers312,10112
Non-polymers2,46851
Water77543
1
C: Autophagy-related protein 38
D: Autophagy-related protein 38
A: Autophagy-related protein 38
B: Autophagy-related protein 38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,81720
Polymers104,0344
Non-polymers78316
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
G: Autophagy-related protein 38
H: Autophagy-related protein 38
E: Autophagy-related protein 38
F: Autophagy-related protein 38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,78920
Polymers104,0344
Non-polymers75616
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
K: Autophagy-related protein 38
L: Autophagy-related protein 38
I: Autophagy-related protein 38
J: Autophagy-related protein 38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,96323
Polymers104,0344
Non-polymers92919
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)81.383, 249.385, 50.197
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

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Protein , 1 types, 12 molecules CDABGHKLEFIJ

#1: Protein
Autophagy-related protein 38


Mass: 26008.424 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ATG38, YLR211C, L8167.20 / Production host: Escherichia coli (E. coli) / References: UniProt: Q05789

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Non-polymers , 8 types, 94 molecules

#2: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Na
#3: Chemical
ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: NO3
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-NH4 / AMMONIUM ION


Mass: 18.038 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: H4N
#7: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#8: Chemical ChemComp-IOD / IODIDE ION


Mass: 126.904 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: I
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 43 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.5 Å3/Da / Density % sol: 65 %
Crystal growTemperature: 290 K / Method: vapor diffusion, sitting drop / pH: 5
Details: 1.97 M ammonium nitrate, 0.15 M (D/L) malate pH 5.0 and 0.136 M Mg sulfate The full-length Atg38 protein was subjected to limited proteolysis. Atg38 (300 microliters at 16 mg/mL in gel- ...Details: 1.97 M ammonium nitrate, 0.15 M (D/L) malate pH 5.0 and 0.136 M Mg sulfate The full-length Atg38 protein was subjected to limited proteolysis. Atg38 (300 microliters at 16 mg/mL in gel-filtration buffer containing 20 mM Tris-HCl pH 8.8, 150 mM NaCl, 1 mM TCEP) was mixed with subtilisin (3 microliters at 1 mg/mL, using subtilisn from the ProteAce kit, Hampton Research), incubated for 10 h at 4 degrees C and set up for crystallization without further purification
Temp details: 290.15

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.97626 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 29, 2014 / Details: channel-cut crystal, the Si(311) monochromator
RadiationMonochromator: channel-cut silicon monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97626 Å / Relative weight: 1
ReflectionResolution: 2.2→58.1 Å / Num. obs: 52821 / % possible obs: 99.8 % / Observed criterion σ(F): -1 / Observed criterion σ(I): -1 / Redundancy: 6.6 % / Biso Wilson estimate: 36 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.062 / Rsym value: 0.062 / Net I/av σ(I): 6 / Net I/σ(I): 16.9
Reflection shellResolution: 2.2→2.27 Å / Redundancy: 6.3 % / Rmerge(I) obs: 0.891 / Mean I/σ(I) obs: 2.3 / % possible all: 99.6

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Processing

Software
NameVersionClassification
PHENIXdev_1810refinement
XDSdata reduction
Aimlessdata scaling
autoSHARPphasing
RefinementMethod to determine structure: MAD / Resolution: 2.2→58 Å / SU ML: 0.23 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.94
RfactorNum. reflection% reflectionSelection details
Rfree0.2406 2529 4.79 %0.24
Rwork0.2046 ---
obs0.2064 52821 99.57 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.2→58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4372 0 136 43 4551
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0054509
X-RAY DIFFRACTIONf_angle_d0.7695971
X-RAY DIFFRACTIONf_dihedral_angle_d11.4231826
X-RAY DIFFRACTIONf_chiral_restr0.028649
X-RAY DIFFRACTIONf_plane_restr0.003769
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.2-2.24230.35621200.3112712X-RAY DIFFRACTION99
2.2423-2.28810.28591390.27612764X-RAY DIFFRACTION99
2.2881-2.33780.33561320.262737X-RAY DIFFRACTION100
2.3378-2.39220.29671250.24142812X-RAY DIFFRACTION99
2.3922-2.45210.2681420.22272717X-RAY DIFFRACTION100
2.4521-2.51830.26461450.19462792X-RAY DIFFRACTION100
2.5183-2.59250.24571410.19652729X-RAY DIFFRACTION100
2.5925-2.67610.24641420.20042762X-RAY DIFFRACTION100
2.6761-2.77180.19491420.19832757X-RAY DIFFRACTION99
2.7718-2.88280.21351390.19412756X-RAY DIFFRACTION99
2.8828-3.01390.21321440.18472792X-RAY DIFFRACTION100
3.0139-3.17280.23321440.19542806X-RAY DIFFRACTION100
3.1728-3.37160.24521410.18762784X-RAY DIFFRACTION100
3.3716-3.63190.19081410.17522800X-RAY DIFFRACTION99
3.6319-3.99730.2211470.18172816X-RAY DIFFRACTION100
3.9973-4.57550.1921450.18052866X-RAY DIFFRACTION100
4.5755-5.76390.2421460.20942863X-RAY DIFFRACTION100
5.7639-58.1740.30841540.24343027X-RAY DIFFRACTION99

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