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- EMDB-8235: Negative stain structure of Vps15/Vps34 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-8235
TitleNegative stain structure of Vps15/Vps34 complex
Map dataVps15/34 complex
Sample
  • Complex: Vps15/34
    • Protein or peptide: Phosphatidylinositol 3-kinase VPS34
    • Protein or peptide: Serine/threonine-protein kinase VPS15
Function / homology
Function and homology information


Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the early endosome membrane / RHO GTPases Activate NADPH Oxidases / autophagy of peroxisome / nucleus-vacuole junction / Synthesis of PIPs at the Golgi membrane / vacuole-isolation membrane contact site / vacuole inheritance / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I ...Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the early endosome membrane / RHO GTPases Activate NADPH Oxidases / autophagy of peroxisome / nucleus-vacuole junction / Synthesis of PIPs at the Golgi membrane / vacuole-isolation membrane contact site / vacuole inheritance / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / Macroautophagy / protein retention in Golgi apparatus / pexophagy / phagophore assembly site membrane / protein targeting to vacuole / late endosome to vacuole transport / fungal-type vacuole membrane / phagophore assembly site / phosphatidylinositol-mediated signaling / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / phosphatidylinositol phosphate biosynthetic process / autophagosome assembly / ubiquitin binding / positive regulation of transcription elongation by RNA polymerase II / macroautophagy / autophagy / peroxisome / endocytosis / protein transport / late endosome / endosome membrane / non-specific serine/threonine protein kinase / endosome / protein kinase activity / phosphorylation / Golgi membrane / protein serine kinase activity / protein serine/threonine kinase activity / mitochondrion / ATP binding / membrane / cytosol / cytoplasm
Similarity search - Function
Serine/threonine-protein kinase Vps15-like / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / Phosphoinositide 3-kinase C2 / Phosphoinositide 3-kinase, region postulated to contain C2 domain / C2 phosphatidylinositol 3-kinase-type domain / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase family, accessory domain (PIK domain) ...Serine/threonine-protein kinase Vps15-like / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / Phosphoinositide 3-kinase C2 / Phosphoinositide 3-kinase, region postulated to contain C2 domain / C2 phosphatidylinositol 3-kinase-type domain / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / C2 domain superfamily / Armadillo-like helical / Armadillo-type fold / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / WD40-repeat-containing domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase VPS15 / Phosphatidylinositol 3-kinase VPS34
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 28.0 Å
AuthorsKirsten ML / Zhang L / Ohashi Y / Perisic O / Williams RL / Sachse C
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, 2016-
Header (metadata) releaseJun 22, 2016-
Map releaseOct 5, 2016-
UpdateNov 13, 2019-
Current statusNov 13, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5kc2
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8235.png

Downloads & links

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Map

FileDownload / File: emd_8235.map.gz / Format: CCP4 / Size: 4.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVps15/34 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.82 Å/pix.
x 104 pix.
= 397.28 Å		3.82 Å/pix.
x 104 pix.
= 397.28 Å		3.82 Å/pix.
x 104 pix.
= 397.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.034498956 - 0.18655433
Average (Standard dev.)-0.0010759196 (±0.009914962)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-52-52-52
Dimensions104104104
Spacing104104104
CellA=B=C: 397.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.823.823.82
M x/y/z104104104
origin x/y/z0.0000.0000.000
length x/y/z397.280397.280397.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS104104104
D min/max/mean-0.0340.187-0.001

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Supplemental data

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Sample components

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Entire : Vps15/34

EntireName: Vps15/34
Components
  • Complex: Vps15/34
    • Protein or peptide: Phosphatidylinositol 3-kinase VPS34
    • Protein or peptide: Serine/threonine-protein kinase VPS15

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Supramolecule #1: Vps15/34

SupramoleculeName: Vps15/34 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Subcomplex of Vps34 phosphatidylinositol 3-kinase (PtdIns3K) complex I (yeast)
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pYO225

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Macromolecule #1: Phosphatidylinositol 3-kinase VPS34

MacromoleculeName: Phosphatidylinositol 3-kinase VPS34 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 101.04718 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSLNNITFCV SQDLDVPLKV KIKSLEGHKP LLKPSQKILN PELMLIGSNV FPSSDLIVSL QVFDKERNRN LTLPIYTPYI PFRNSRTWD YWLTLPIRIK QLTFSSHLRI ILWEYNGSKQ IPFFNLETSI FNLKDCTLKR GFESLKFRYD VIDHCEVVTD N KDQENLNK ...String:
MSLNNITFCV SQDLDVPLKV KIKSLEGHKP LLKPSQKILN PELMLIGSNV FPSSDLIVSL QVFDKERNRN LTLPIYTPYI PFRNSRTWD YWLTLPIRIK QLTFSSHLRI ILWEYNGSKQ IPFFNLETSI FNLKDCTLKR GFESLKFRYD VIDHCEVVTD N KDQENLNK YFQGEFTRLP WLDEITISKL RKQRENRTWP QGTFVLNLEF PMLELPVVFI EREIMNTQMN IPTLKNNPGL ST DLREPNR NDPQIKISLG DKYHSTLKFY DPDQPNNDPI EEKYRRLERA SKNANLDKQV KPDIKKRDYL NKIINYPPGT KLT AHEKGS IWKYRYYLMN NKKALTKLLQ STNLREESER VEVLELMDSW AEIDIDDALE LLGSTFKNLS VRSYAVNRLK KASD KELEL YLLQLVEAVC FENLSTFSDK SNSEFTIVDA VSSQKLSGDS MLLSTSHANQ KLLKSISSES ETSGTESLPI VISPL AEFL IRRALVNPRL GSFFYWYLKS ESEDKPYLDQ ILSSFWSRLD KKSRNILNDQ VRLINVLREC CETIKRLKDT TAKKME LLV HLLETKVRPL VKVRPIALPL DPDVLICDVC PETSKVFKSS LSPLKITFKT TLNQPYHLMF KVGDDLRQDQ LVVQIIS LM NELLKNENVD LKLTPYKILA TGPQEGAIEF IPNDTLASIL SKYHGILGYL KLHYPDENAT LGVQGWVLDN FVKSCAGY C VITYILGVGD RHLDNLLVTP DGHFFHADFG YILGQDPKPF PPLMKLPPQI IEAFGGAESS NYDKFRSYCF VAYSILRRN AGLILNLFEL MKTSNIPDIR IDPNGAILRV RERFNLNMSE EDATVHFQNL INDSVNALLP IVIDHLHNLA QYWRT

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Macromolecule #2: Serine/threonine-protein kinase VPS15

MacromoleculeName: Serine/threonine-protein kinase VPS15 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 166.55 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGAQLSLVVQ ASPSIAIFSY IDVLEEVHYV SQLNSSRFLK TCKALDPNGE IVIKVFIKPK DQYSLRPFLQ RIRAQSFKLG QLPHVLNYS KLIETNRAGY MIRQHLKNNL YDRLSLRPYL QDIELKFIAF QLLNALKDIH NLNIVHGDIK TENILVTSWN W CILTDFAA ...String:
MGAQLSLVVQ ASPSIAIFSY IDVLEEVHYV SQLNSSRFLK TCKALDPNGE IVIKVFIKPK DQYSLRPFLQ RIRAQSFKLG QLPHVLNYS KLIETNRAGY MIRQHLKNNL YDRLSLRPYL QDIELKFIAF QLLNALKDIH NLNIVHGDIK TENILVTSWN W CILTDFAA FIKPVYLPED NPGEFLFYFD TSKRRTCYLA PERFNSKLYQ DGKSNNGRLT KEMDIFSLGC VIAEIFAEGR PI FNLSQLF KYKSNSYDVN REFLMEEMNS TDLRNLVLDM IQLDPSKRLS CDELLNKYRG IFFPDYFYTF IYDYFRNLVT MTT STPISD NTCTNSTLED NVKLLDETTE KIYRDFSQIC HCLDFPLIKD GGEIGSDPPI LESYKIEIEI SRFLNTNLYF PQNY HLVLQ QFTKVSEKIK SVKEECALLF ISYLSHSIRS IVSTATKLKN LELLAVFAQF VSDENKIDRV VPYFVCCFED SDQDV QALS LLTLIQVLTS VRKLNQLNEN IFVDYLLPRL KRLLISNRQN TNYLRIVFAN CLSDLAIIIN RFQEFTFAQH CNDNSM DNN TEIMESSTKY SAKLIQSVED LTVSFLTDND TYVKMALLQN ILPLCKFFGR ERTNDIILSH LITYLNDKDP ALRVSLI QT ISGISILLGT VTLEQYILPL LIQTITDSEE LVVISVLQSL KSLFKTGLIR KKYYIDISKT TSPLLLHPNN WIRQFTLM I IIEIINKLSK AEVYCILYPI IRPFFEFDVE FNFKSMISCC KQPVSRSVYN LLCSWSVRAS KSLFWKKIIT NHVDSFGNN RIEFITKNYS SKNYGFNKRD TKSSSSLKGI KTSSTVYSHD NKEIPLTAED RNWIDKFHII GLTEKDIWKI VALRGYVIRT ARVMAANPD FPYNNSNYRP LVQNSPPNLN LTNIMPRNIF FDVEFAEEST SEGQDSNLEN QQIYKYDESE KDSNKLNING S KQLSTVMD INGSLIFKNK SIATTTSNLK NVFVQLEPTS YHMHSPNHGL KDNANVKPER KVVVSNSYEG DVESIEKFLS TF KILPPLR DYKEFGPIQE IVRSPNMGNL RGKLIATLME NEPNSITSSA VSPGETPYLI TGSDQGVIKI WNLKEIIVGE VYS SSLTYD CSSTVTQITM IPNFDAFAVS SKDGQIIVLK VNHYQQESEV KFLNCECIRK INLKNFGKNE YAVRMRAFVN EEKS LLVAL TNLSRVIIFD IRTLERLQII ENSPRHGAVS SICIDEECCV LILGTTRGII DIWDIRFNVL IRSWSFGDHA PITHV EVCQ FYGKNSVIVV GGSSKTFLTI WNFVKGHCQY AFINSDEQPS MEHFLPIEKG LEELNFCGIR SLNALSTISV SNDKIL LTD EATSSIVMFS LNELSSSKAV ISPSRFSDVF IPTQVTANLT MLLRKMKRTS THSVDDSLYH HDIINSISTC EVDETPL LV ACDNSGLIGI FQ

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 8.8
Component:
ConcentrationName
20.0 mMTris
500.0 mMNaClSodium chloride
1.0 %CHAPS
2.0 mMDTT
StainingType: NEGATIVE / Material: Uranyl acetate / Details: droplet technique
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 2 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 14172
CTF correctionSoftware - Name: EMAN (ver. 2)
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 27 / Random conical tilt - Tilt angle: 55 degrees
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER
Final 3D classificationSoftware - Name: IMAGIC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 14172

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