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Yorodumi- EMDB-12238: Sample bin4 tomogram for the VPS34 complex II on Rab5a coupled li... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12238 | ||||||||||||||||||||||||||||||
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Title | Sample bin4 tomogram for the VPS34 complex II on Rab5a coupled lipid vesicles | ||||||||||||||||||||||||||||||
Map data | Sample tomogram before ctf correction. This is one of the tomograms used in END-12214 subtomogram average reconstruction. | ||||||||||||||||||||||||||||||
Sample |
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Biological species | Homo sapiens (human) | ||||||||||||||||||||||||||||||
Method | electron tomography / cryo EM | ||||||||||||||||||||||||||||||
Authors | Tremel S / Ohashi Y / Morado DR / Bertram J / Perisic O / Brandt LTL / von Wrisberg M-K / Chen ZA / Maslen SL / Kovtun O ...Tremel S / Ohashi Y / Morado DR / Bertram J / Perisic O / Brandt LTL / von Wrisberg M-K / Chen ZA / Maslen SL / Kovtun O / Skehel M / Rappsilber J / Lang K / Munro S / Briggs JAG / Williams RL | ||||||||||||||||||||||||||||||
Funding support | United Kingdom, Germany, 9 items
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Citation | Journal: Nat Commun / Year: 2021 Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes. Authors: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel ...Authors: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel / Juri Rappsilber / Kathrin Lang / Sean Munro / John A G Briggs / Roger L Williams / Abstract: The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal ...The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations. | ||||||||||||||||||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12238.map.gz | 1.3 GB | EMDB map data format | |
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Header (meta data) | emd-12238-v30.xml emd-12238.xml | 22.5 KB 22.5 KB | Display Display | EMDB header |
Images | emd_12238.png | 67.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12238 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12238 | HTTPS FTP |
-Validation report
Summary document | emd_12238_validation.pdf.gz | 143.7 KB | Display | EMDB validaton report |
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Full document | emd_12238_full_validation.pdf.gz | 142.8 KB | Display | |
Data in XML | emd_12238_validation.xml.gz | 5 KB | Display | |
Data in CIF | emd_12238_validation.cif.gz | 5.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12238 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12238 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_12238.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Sample tomogram before ctf correction. This is one of the tomograms used in END-12214 subtomogram average reconstruction. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 8.532 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...
Entire | Name: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a on lipid vesicles |
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Components |
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-Supramolecule #1: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...
Supramolecule | Name: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a on lipid vesicles type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 ...Details: The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 (encoding UVGRAG 1-464 fused to the BATS of ATG14, residues 413-492 in pVAG) and pYO1006 (Beclin1 in pCAG) |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 392.16 KDa |
-Macromolecule #1: UV radiation resistance-associated gene protein
Macromolecule | Name: UV radiation resistance-associated gene protein / type: protein_or_peptide / ID: 1 / Details: Human UVRAG / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MSASASVGGP VPQPPPGPAA ALPPGSAARA LHVELPSQQR RLRHLRNIAA RNIVNRNGHQ LLDTYFTLHL CSTEKIYKEF YRSEVIKNS LNPTWRSLDF GIMPDRLDTS VSCFVVKIWG GKENIYQLLI EWKVCLDGLK YLGQQIHARN QNEIIFGLND G YYGAPFEH ...String: MSASASVGGP VPQPPPGPAA ALPPGSAARA LHVELPSQQR RLRHLRNIAA RNIVNRNGHQ LLDTYFTLHL CSTEKIYKEF YRSEVIKNS LNPTWRSLDF GIMPDRLDTS VSCFVVKIWG GKENIYQLLI EWKVCLDGLK YLGQQIHARN QNEIIFGLND G YYGAPFEH KGYSNAQKTI LLQVDQNCVR NSYDVFSLLR LHRAQCAIKQ TQVTVQKIGK EIEEKLRLTS TSNELKKKSE CL QLKILVL QNELERQKKA LGREVALLHK QQIALQDKGS AFSAEHLKLQ LQKESLNELR KECTAKRELF LKTNAQLTIR CRQ LLSELS YIYPIDLNEH KDYFVCGVKL PNSEDFQAKD DGSIAVALGY TAHLVSMISF FLQVPLRYPI IHKGSRSTIK DNIN DKLTE KEREFPLYPK GGEKLQFDYG VYLLNKNIAQ LRYQHGLGTP DLRQTLPNLK NFMEHGLMVR CDRHHTSSAI PVPKR QSSI FGGADVGFSG GIPSPDKGHR KRASSENERL QYKTPPPSYN SALAQPVTTV PSMGETERKI TSLSSSLDTS LDFSKE NKK KGEDLVGSLN GGHANVHPSQ EQGEALSGHR ATVNGTLLPS EQAGSASVQL PGEFHPVSEA ELCCTVEQAE EIIGLEA TG FASGDQLEAF NCIPVDSAVA VECDEQVLGE FEEFSRRIYA LNENVSSFRR PRRSSDK |
-Macromolecule #2: Phosphatidylinositol 3-kinase catalytic subunit type 3
Macromolecule | Name: Phosphatidylinositol 3-kinase catalytic subunit type 3 type: protein_or_peptide / ID: 2 / Details: human vps34 / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MGEAEKFHYI YSCDLDINVQ LKIGSLEGKR EQKSYKAVLE DPMLKFSGLY QETCSDLYVT CQVFAEGKPL ALPVRTSYKA FSTRWNWNE WLKLPVKYPD LPRNAQVALT IWDVYGPGKA VPVGGTTVSL FGKYGMFRQG MHDLKVWPNV EADGSEPTKT P GRTSSTLS ...String: MGEAEKFHYI YSCDLDINVQ LKIGSLEGKR EQKSYKAVLE DPMLKFSGLY QETCSDLYVT CQVFAEGKPL ALPVRTSYKA FSTRWNWNE WLKLPVKYPD LPRNAQVALT IWDVYGPGKA VPVGGTTVSL FGKYGMFRQG MHDLKVWPNV EADGSEPTKT P GRTSSTLS EDQMSRLAKL TKAHRQGHMV KVDWLDRLTF REIEMINESE KRSSNFMYLM VEFRCVKCDD KEYGIVYYEK DG DESSPIL TSFELVKVPD PQMSMENLVE SKHHKLARSL RSGPSDHDLK PNAATRDQLN IIVSYPPTKQ LTYEEQDLVW KFR YYLTNQ EKALTKFLKC VNWDLPQEAK QALELLGKWK PMDVEDSLEL LSSHYTNPTV RRYAVARLRQ ADDEDLLMYL LQLV QALKY ENFDDIKNGL EPTKKDSQSS VSENVSNSGI NSAEIDSSQI ITSPLPSVSS PPPASKTKEV PDGENLEQDL CTFLI SRAC KNSTLANYLY WYVIVECEDQ DTQQRDPKTH EMYLNVMRRF SQALLKGDKS VRVMRSLLAA QQTFVDRLVH LMKAVQ RES GNRKKKNERL QALLGDNEKM NLSDVELIPL PLEPQVKIRG IIPETATLFK SALMPAQLFF KTEDGGKYPV IFKHGDD LR QDQLILQIIS LMDKLLRKEN LDLKLTPYKV LATSTKHGFM QFIQSVPVAE VLDTEGSIQN FFRKYAPSEN GPNGISAE V MDTYVKSCAG YCVITYILGV GDRHLDNLLL TKTGKLFHID FGYILGRDPK PLPPPMKLNK EMVEGMGGTQ SEQYQEFRK QCYTAFLHLR RYSNLILNLF SLMVDANIPD IALEPDKTVK KVQDKFRLDL SDEEAVHYMQ SLIDESVHAL FAAVVEQIHK FAQYWRK |
-Macromolecule #3: Phosphoinositide 3-kinase regulatory subunit 4
Macromolecule | Name: Phosphoinositide 3-kinase regulatory subunit 4 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MGNQLAGIAP SQILSVESYF SDIHDFEYDK SLGSTRFFKV ARAKHREGLV VVKVFAIQDP TLPLTSYKQE LEELKIRLNS AQNCLPFQK ASEKASEKAA MLFRQYVRDN LYDRISTRPF LNNIEKRWIA FQILTAVDQA HKSGVRHGDI KTENVMVTSW N WVLLTDFA ...String: MGNQLAGIAP SQILSVESYF SDIHDFEYDK SLGSTRFFKV ARAKHREGLV VVKVFAIQDP TLPLTSYKQE LEELKIRLNS AQNCLPFQK ASEKASEKAA MLFRQYVRDN LYDRISTRPF LNNIEKRWIA FQILTAVDQA HKSGVRHGDI KTENVMVTSW N WVLLTDFA SFKPTYLPED NPADFNYFFD TSRRRTCYIA PERFVDGGMF ATELEYMRDP STPLVDLNSN QRTRGELKRA MD IFSAGCV IAELFTEGVP LFDLSQLLAY RNGHFFPEQV LNKIEDHSIR ELVTQMIHRE PDKRLEAEDY LKQQRGNAFP EIF YTFLQP YMAQFAKETF LSADERILVI RKDLGNIIHN LCGHDLPEKA EGEPKENGLV ILVSVITSCL QTLKYCDSKL AALE LILHL APRLSVEILL DRITPYLLHF SNDSVPRVRA EALRTLTKVL ALVKEVPRND INIYPEYILP GIAHLAQDDA TIVRL AYAE NIALLAETAL RFLELVQLKN LNMENDPNNE EIDEVTHPNG NYDTELQALH EMVQQKVVTL LSDPENIVKQ TLMENG ITR LCVFFGRQKA NDVLLSHMIT FLNDKNDWHL RGAFFDSIVG VAAYVGWQSS SILKPLLQQG LSDAEEFVIV KALYALT CM CQLGLLQKPH VYEFASDIAP FLCHPNLWIR YGAVGFITVV ARQISTADVY CKLMPYLDPY ITQPIIQIER KLVLLSVL K EPVSRSIFDY ALRSKDITSL FRHLHMRQKK RNGSLPDCPP PEDPAIAQLL KKLLSQGMTE EEEDKLLALK DFMMKSNKA KANIVDQSHL HDSSQKGVID LAALGITGRQ VDLVKTKQEP DDKRARKHVK QDSNVNEEWK SMFGSLDPPN MPQALPKGSD QEVIQTGKP PRSESSAGIC VPLSTSSQVP EVTTVQNKKP VIPVLSSTIL PSTYQIRITT CKTELQQLIQ QKREQCNAER I AKQMMENA EWESKPPPPG WRPKGLLVAH LHEHKSAVNR IRVSDEHSLF ATCSNDGTVK IWNSQKMEGK TTTTRSILTY SR IGGRVKT LTFCQGSHYL AIASDNGAVQ LLGIEASKLP KSPKIHPLQS RILDQKEDGC VVDMHHFNSG AQSVLAYATV NGS LVGWDL RSSSNAWTLK HDLKSGLITS FAVDIHQCWL CIGTSSGTMA CWDMRFQLPI SSHCHPSRAR IRRLSMHPLY QSWV IAAVQ GNNEVSMWDM ETGDRRFTLW ASSAPPLSEL QPSPHSVHGI YCSPADGNPI LLTAGSDMKI RFWDLAYPER SYVVA GSTS SPSVSYYRKI IEGTEVVQEI QNKQKVGPSD DTPRRGPESL PVGHHDIITD VATFQTTQGF IVTASRDGIV KVWKSR PTT ASENLYFQ |
-Macromolecule #4: Beclin-1
Macromolecule | Name: Beclin-1 / type: protein_or_peptide / ID: 4 / Details: Beclin1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MEGSKTSNNS TMQVSFVCQR CSQPLKLDTS FKILDRVTIQ ELTAPLLTTA QAKPGETQEE ETNSGEEPFI ETPRQDGVSR RFIPPARMM STESANSFTL IGEASDGGTM ENLSRRLKVT GDLFDIMSGQ TDVDHPLCEE CTDTLLDQLD TQLNVTENEC Q NYKRCLEI ...String: MEGSKTSNNS TMQVSFVCQR CSQPLKLDTS FKILDRVTIQ ELTAPLLTTA QAKPGETQEE ETNSGEEPFI ETPRQDGVSR RFIPPARMM STESANSFTL IGEASDGGTM ENLSRRLKVT GDLFDIMSGQ TDVDHPLCEE CTDTLLDQLD TQLNVTENEC Q NYKRCLEI LEQMNEDDSE QLQMELKELA LEEERLIQEL EDVEKNRKIV AENLEKVQAE AERLDQEEAQ YQREYSEFKR QQ LELDDEL KSVENQMRYA QTQLDKLKKT NVFNATFHIW HSGQFGTINN FRLGRLPSVP VEWNEINAAW GQTVLLLHAL ANK MGLKFQ RYRLVPYGNH SYLESLTDKS KELPLYCSGG LRFFWDNKFD HAMVAFLDCV QQFKEEVEKG ETRFCLPYRM DVEK GKIED TGGSGGSYSI KTQFNSEEQW TKALKFMLTN LKWGLAWVSS QFYNK |
-Macromolecule #5: Ras-related protein Rab-5A
Macromolecule | Name: Ras-related protein Rab-5A / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO / EC number: small monomeric GTPase |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: NKISQFKLVL LGESAVGKSS LVLRFVKGQF HEFQESTIGA AFLTQTVSLD DTTVKFEIWD TAGLERYHSL APMYYRGAQA AIVVYDITN EESFARAKNW VKELQRQASP NIVIALSGNK ADLANKRAVD FQEAQSYADD NSLLFMETSA KTSMNVNEIF M AIAKKLPK |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | 3D array |
-Sample preparation
Concentration | 6.2 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: Quorum SC7620 | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 316 K / Instrument: FEI VITROBOT MARK I / Details: blot force was 20, with a blot time of 6 s. | ||||||||||||
Details | Large unilamellar lipid vesicles containing lipid-modified Rab5A to which VPS34 complex II is bound. 50 mM HEPES, 200 mM NaCl, 0.5 mM TCEP, 6 mg/ml lipid vesicles. | ||||||||||||
Cryo protectant | None | ||||||||||||
Sectioning | Other: NO SECTIONING | ||||||||||||
Fiducial marker | Manufacturer: BBI Solutions EM / Diameter: 10 nm |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Sampling interval: 2.133 µm / Number grids imaged: 1 / Average exposure time: 0.55 sec. / Average electron dose: 2.99 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: IMOD (ver. 4.10) / Number images used: 41 |
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CTF correction | Software - Name: NOVACTF |