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- EMDB-26734: Cryo-EM structure of WAVE regulatory complex with Rac1 bound on b... -

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Basic information

Entry
Database: EMDB / ID: EMD-26734
TitleCryo-EM structure of WAVE regulatory complex with Rac1 bound on both A and D site
Map dataWRC230VCA-2Rac1 complex sharpened map
Sample
  • Complex: WAVE regulatory complex with Rac1 bound to A and D sites
    • Protein or peptide: Cytoplasmic FMR1-interacting protein 1
    • Protein or peptide: Nck-associated protein 1
    • Protein or peptide: Wiskott-Aldrich syndrome protein family member 1
    • Protein or peptide: Protein BRICK1
    • Protein or peptide: Abl interactor 2
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
Function / homology
Function and homology information


peripheral region of growth cone / negative regulation of synaptic vesicle recycling / intracellular protein-containing complex / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of synaptic structure / regulation of actin polymerization or depolymerization ...peripheral region of growth cone / negative regulation of synaptic vesicle recycling / intracellular protein-containing complex / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of synaptic structure / regulation of actin polymerization or depolymerization / regulation of respiratory burst / central region of growth cone / modification of postsynaptic actin cytoskeleton / negative regulation of interleukin-23 production / dendrite extension / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / regulation of translation at postsynapse, modulating synaptic transmission / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / engulfment of apoptotic cell / Inactivation of CDC42 and RAC1 / filopodium tip / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / : / regulation of modification of postsynaptic actin cytoskeleton / cortical cytoskeleton organization / respiratory burst / regulation of actin filament polymerization / hepatocyte growth factor receptor signaling pathway / RNA 7-methylguanosine cap binding / ruffle organization / cell projection assembly / thioesterase binding / negative regulation of fibroblast migration / regulation of stress fiber assembly / RHO GTPases activate CIT / Nef and signal transduction / axon extension / sphingosine-1-phosphate receptor signaling pathway / PCP/CE pathway / motor neuron axon guidance / RHO GTPases activate KTN1 / regulation of nitric oxide biosynthetic process / regulation of lamellipodium assembly / positive regulation of neutrophil chemotaxis / Azathioprine ADME / Activation of RAC1 / positive regulation of ruffle assembly / positive regulation of dendrite development / DCC mediated attractive signaling / positive regulation of cell-substrate adhesion / MET activates RAP1 and RAC1 / Wnt signaling pathway, planar cell polarity pathway / Sema4D mediated inhibition of cell attachment and migration / CD28 dependent Vav1 pathway / Ephrin signaling / protein kinase A binding / lamellipodium assembly / regulation of myelination / cortical actin cytoskeleton organization / positive regulation of Rho protein signal transduction / establishment or maintenance of cell polarity / regulation of cell size / DSCAM interactions / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / Rho GDP-dissociation inhibitor binding / positive regulation of actin filament polymerization / NRAGE signals death through JNK / Rac protein signal transduction / protein kinase A regulatory subunit binding / filamentous actin / RHO GTPases activate PAKs / dendritic growth cone / positive regulation of focal adhesion assembly / lamellipodium membrane / semaphorin-plexin signaling pathway / excitatory synapse / ficolin-1-rich granule membrane / Sema3A PAK dependent Axon repulsion / RHOG GTPase cycle / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate NADPH Oxidases / RHO GTPases Activate WASPs and WAVEs / anatomical structure morphogenesis / RAC3 GTPase cycle / RAC2 GTPase cycle / RHO GTPases activate IQGAPs / positive regulation of axon extension / localization / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly / axonal growth cone / response to electrical stimulus
Similarity search - Function
Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / CYRIA/CYRIB, Rac1 binding domain / Abl-interactor, homeo-domain homologous domain / SCAR/WAVE family / ABI family ...Cytoplasmic FMR1-interacting / Nck-associated protein 1 / Cytoplasmic Fragile-X interacting family / Nck-associated protein 1 / Protein BRICK1 / Abl interactor 2, SH3 domain / CYRIA/CYRIB, Rac1 binding domain / Abl-interactor, homeo-domain homologous domain / SCAR/WAVE family / ABI family / CYRIA/CYRIB Rac1 binding domain / Abl-interactor HHR / WH2 motif / Wiskott Aldrich syndrome homology region 2 / WH2 domain / WH2 domain profile. / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Small GTPase Rho / small GTPase Rho family profile. / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / SH3 domain / Small GTPase / Ras family / Rab subfamily of small GTPases / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Abl interactor 2 / Ras-related C3 botulinum toxin substrate 1 / Cytoplasmic FMR1-interacting protein 1 / Protein BRICK1 / Actin-binding protein WASF1 / Nck-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsDing B / Yang S / Chen B / Chowdhury S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2022
Title: Structures reveal a key mechanism of WAVE regulatory complex activation by Rac1 GTPase.
Authors: Bojian Ding / Sheng Yang / Matthias Schaks / Yijun Liu / Abbigale J Brown / Klemens Rottner / Saikat Chowdhury / Baoyu Chen /
Abstract: The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the ...The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.
History
DepositionApr 25, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateNov 9, 2022-
Current statusNov 9, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26734.png

Downloads & links

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Map

FileDownload / File: emd_26734.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationWRC230VCA-2Rac1 complex sharpened map
Voxel sizeX=Y=Z: 0.8757 Å
Density
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.008826853 - 1.5363575
Average (Standard dev.)0.0074820793 (±0.055648733)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 224.1792 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26734_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: WRC230VCA-2Rac1 complex masked unsharpened map

Fileemd_26734_additional_1.map
AnnotationWRC230VCA-2Rac1 complex masked unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: WRC230VCA-2Rac1 complex unfiltered unmasked unsharpened first half map...

Fileemd_26734_half_map_1.map
AnnotationWRC230VCA-2Rac1 complex unfiltered unmasked unsharpened first half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: WRC230VCA-2Rac1 complex unfiltered unmasked unsharpened second half map...

Fileemd_26734_half_map_2.map
AnnotationWRC230VCA-2Rac1 complex unfiltered unmasked unsharpened second half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : WAVE regulatory complex with Rac1 bound to A and D sites

EntireName: WAVE regulatory complex with Rac1 bound to A and D sites
Components
  • Complex: WAVE regulatory complex with Rac1 bound to A and D sites
    • Protein or peptide: Cytoplasmic FMR1-interacting protein 1
    • Protein or peptide: Nck-associated protein 1
    • Protein or peptide: Wiskott-Aldrich syndrome protein family member 1
    • Protein or peptide: Protein BRICK1
    • Protein or peptide: Abl interactor 2
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
    • Protein or peptide: Ras-related C3 botulinum toxin substrate 1
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate

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Supramolecule #1: WAVE regulatory complex with Rac1 bound to A and D sites

SupramoleculeName: WAVE regulatory complex with Rac1 bound to A and D sites
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 380 KDa

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Macromolecule #1: Cytoplasmic FMR1-interacting protein 1

MacromoleculeName: Cytoplasmic FMR1-interacting protein 1 / type: protein_or_peptide / ID: 1
Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 145.36375 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAAQVTLEDA LSNVDLLEEL PLPDQQPCIE PPPSSLLYQP NFNTNFEDRN AFVTGIARYI EQATVHSSMN EMLEEGQEYA VMLYTWRSC SRAIPQVKCN EQPNRVEIYE KTVEVLEPEV TKLMNFMYFQ RNAIERFCGE VRRLCHAERR KDFVSEAYLI T LGKFINMF ...String:
MAAQVTLEDA LSNVDLLEEL PLPDQQPCIE PPPSSLLYQP NFNTNFEDRN AFVTGIARYI EQATVHSSMN EMLEEGQEYA VMLYTWRSC SRAIPQVKCN EQPNRVEIYE KTVEVLEPEV TKLMNFMYFQ RNAIERFCGE VRRLCHAERR KDFVSEAYLI T LGKFINMF AVLDELKNMK CSVKNDHSAY KRAAQFLRKM ADPQSIQESQ NLSMFLANHN KITQSLQQQL EVISGYEELL AD IVNLCVD YYENRMYLTP SEKHMLLKVM GFGLYLMDGS VSNIYKLDAK KRINLSKIDK YFKQLQVVPL FGDMQIELAR YIK TSAHYE ENKSRWTCTS SGSSPQYNIC EQMIQIREDH MRFISELARY SNSEVVTGSG RQEAQKTDAE YRKLFDLALQ GLQL LSQWS AHVMEVYSWK LVHPTDKYSN KDCPDSAEEY ERATRYNYTS EEKFALVEVI AMIKGLQVLM GRMESVFNHA IRHTV YAAL QDFSQVTLRE PLRQAIKKKK NVIQSVLQAI RKTVCDWETG HEPFNDPALR GEKDPKSGFD IKVPRRAVGP SSTQLY MVR TMLESLIADK SGSKKTLRSS LEGPTILDIE KFHRESFFYT HLINFSETLQ QCCDLSQLWF REFFLELTMG RRIQFPI EM SMPWILTDHI LETKEASMME YVLYSLDLYN DSAHYALTRF NKQFLYDEIE AEVNLCFDQF VYKLADQIFA YYKVMAGS L LLDKRLRSEC KNQGATIHLP PSNRYETLLK QRHVQLLGRS IDLNRLITQR VSAAMYKSLE LAIGRFESED LTSIVELDG LLEINRMTHK LLSRYLTLDG FDAMFREANH NVSAPYGRIT LHVFWELNYD FLPNYCYNGS TNRFVRTVLP FSQEFQRDKQ PNAQPQYLH GSKALNLAYS SIYGSYRNFV GPPHFQVICR LLGYQGIAVV MEELLKVVKS LLQGTILQYV KTLMEVMPKI C RLPRHEYG SPGILEFFHH QLKDIVEYAE LKTVCFQNLR EVGNAILFCL LIEQSLSLEE VCDLLHAAPF QNILPRVHVK EG ERLDAKM KRLESKYAPL HLVPLIERLG TPQQIAIARE GDLLTKERLC CGLSMFEVIL TRIRSFLDDP IWRGPLPSNG VMH VDECVE FHRLWSAMQF VYCIPVGTHE FTVEQCFGDG LHWAGCMIIV LLGQQRRFAV LDFCYHLLKV QKHDGKDEII KNVP LKKMV ERIRKFQILN DEIITILDKY LKSGDGEGTP VEHVRCFQPP IHQSLASS

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Macromolecule #2: Nck-associated protein 1

MacromoleculeName: Nck-associated protein 1 / type: protein_or_peptide / ID: 2
Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 128.940727 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSRSVLQPSQ QKLAEKLTIL NDRGVGMLTR LYNIKKACGD PKAKPSYLID KNLESAVKFI VRKFPAVETR NNNQQLAQLQ KEKSEILKN LALYYFTFVD VMEFKDHVCE LLNTIDVCQV FFDITVNFDL TKNYLDLIIT YTTLMILLSR IEERKAIIGL Y NYAHEMTH ...String:
MSRSVLQPSQ QKLAEKLTIL NDRGVGMLTR LYNIKKACGD PKAKPSYLID KNLESAVKFI VRKFPAVETR NNNQQLAQLQ KEKSEILKN LALYYFTFVD VMEFKDHVCE LLNTIDVCQV FFDITVNFDL TKNYLDLIIT YTTLMILLSR IEERKAIIGL Y NYAHEMTH GASDREYPRL GQMIVDYENP LKKMMEEFVP HSKSLSDALI SLQMVYPRRN LSADQWRNAQ LLSLISAPST ML NPAQSDT MPCEYLSLDA MEKWIIFGFI LCHGILNTDA TALNLWKLAL QSSSCLSLFR DEVFHIHKAA EDLFVNIRGY NKR INDIRE CKEAAVSHAG SMHRERRKFL RSALKELATV LSDQPGLLGP KALFVFMALS FARDEIIWLL RHADNMPKKS ADDF IDKHI AELIFYMEEL RAHVRKYGPV MQRYYVQYLS GFDAVVLNEL VQNLSVCPED ESIIMSSFVN TMTSLSVKQV EDGEV FDFR GMRLDWFRLQ AYTSVSKASL GLADHRELGK MMNTIIFHTK MVDSLVEMLV ETSDLSIFCF YSRAFEKMFQ QCLELP SQS RYSIAFPLLC THFMSCTHEL CPEERHHIGD RSLSLCNMFL DEMAKQARNL ITDICTEQCT LSDQLLPKHC AKTISQA VN KKSKKQTGKK GEPEREKPGV ESMRKNRLVV TNLDKLHTAL SELCFSINYV PNMVVWEHTF TPREYLTSHL EIRFTKSI V GMTMYNQATQ EIAKPSELLT SVRAYMTVLQ SIENYVQIDI TRVFNNVLLQ QTQHLDSHGE PTITSLYTNW YLETLLRQV SNGHIAYFPA MKAFVNLPTE NELTFNAEEY SDISEMRSLS ELLGPYGMKF LSESLMWHIS SQVAELKKLV VENVDVLTQM RTSFDKPDQ MAALFKRLSS VDSVLKRMTI IGVILSFRSL AQEALRDVLS YHIPFLVSSI EDFKDHIPRE TDMKVAMNVY E LSSAAGLP CEIDPALVVA LSSQKSENIS PEEEYKIACL LMVFVAVSLP TLASNVMSQY SPAIEGHCNN IHCLAKAINQ IA AALFTIH KGSIEDRLKE FLALASSSLL KIGQETDKTT TRNRESVYLL LDMIVQESPF LTMDLLESCF PYVLLRNAYH AVY KQSVTS SA

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Macromolecule #3: Wiskott-Aldrich syndrome protein family member 1

MacromoleculeName: Wiskott-Aldrich syndrome protein family member 1 / type: protein_or_peptide / ID: 3
Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification ...Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.009406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPLVKRNIDP RHLCHTALPR GIKNELECVT NISLANIIRQ LSSLSKYAED IFGELFNEAH SFSFRVNSLQ ERVDRLSVSV TQLDPKEEE LSLQDITMRK AFRSSTIQDQ QLFDRKTLPI PLQETYDVCE QPPPLNILTP YRDDGKEGLK FYTNPSYFFD L WKEKMLQD ...String:
MPLVKRNIDP RHLCHTALPR GIKNELECVT NISLANIIRQ LSSLSKYAED IFGELFNEAH SFSFRVNSLQ ERVDRLSVSV TQLDPKEEE LSLQDITMRK AFRSSTIQDQ QLFDRKTLPI PLQETYDVCE QPPPLNILTP YRDDGKEGLK FYTNPSYFFD L WKEKMLQD TEDKRKEKRK QKQKNLDRPH EPEKVPRAPH DRRREWQKLA QGPELAEDDA NLLHKHIEVA NGGGSGGSGG SG GSGGSGG SKRHPSTLPV ISDARSVLLE AIRKGIQLRK VEEQREQEAK HERIENDVAT ILSRRIAVEY SDSEDDSEFD EVD WLE

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Macromolecule #4: Protein BRICK1

MacromoleculeName: Protein BRICK1 / type: protein_or_peptide / ID: 4
Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were ...Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.756915 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAGQEDPVQR EIHQDWANRE YIEIITSSIK KIADFLNSFD MSCRSRLATL NEKLTALERR IEYIEARVTK GETLT

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Macromolecule #5: Abl interactor 2

MacromoleculeName: Abl interactor 2 / type: protein_or_peptide / ID: 5
Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these ...Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.041482 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAELQMLLEE EIPGGRRALF DSYTNLERVA DYCENNYIQS ADKQRALEET KAYTTQSLAS VAYLINTLAN NVLQMLDIQA SQLRRMESS INHISQTVDI HKEKVARREI GILTTNKNTS RTHKIIAPAN LERPVRYIRK PIDYTILDDI GHGVKVSTQ

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Macromolecule #6: Ras-related C3 botulinum toxin substrate 1

MacromoleculeName: Ras-related C3 botulinum toxin substrate 1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.025459 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MQAIKCVVVG DGAVGKTCLL ISYTTNAFSG EYIPTVFDNY SANVMVDGKP VNLGLWDTAG QEDYDRLRPL SYPQTDVFLI CFSLVSPAS FENVRAKWYP EVRHHCPNTP IILVGTKLDL RDDKDTIEKL KEKKLTPITY PQGLAMAKEI GAVKYLECSA L TQRGLKTV FDEAIRAVLC PPPVKKRKRK

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Macromolecule #7: Ras-related C3 botulinum toxin substrate 1

MacromoleculeName: Ras-related C3 botulinum toxin substrate 1 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.010486 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MQAIKCVVVG DGAVGKTCLL ISYTTNAFSG EYIPTVFDNY SANVMVDGKP VNLGLWDTAG LEDYDRLRPL SYPQTDVFLI CFSLVSPAS FENVRAKWYP EVRHHCPNTP IILVGTKLDL RDDKDTIEKL KEKKLTPITY PQGLAMAKEI GAVKYLECSA L TQRGLKTV FDEAIRAVLC PPPVKKRKRK

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Macromolecule #8: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / type: ligand / ID: 8 / Number of copies: 1 / Formula: GNP
Molecular weightTheoretical: 522.196 Da
Chemical component information

ChemComp-GNP:
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GppNHp, GMPPNP, energy-carrying molecule analogue*YM / 5'-Guanylyl imidodiphosphate

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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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

Concentration0.15 mg/mL
BufferpH: 7
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.019 kPa / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsData were collected by shifting the stage to target exposure positions.
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 1285 / Average exposure time: 40.0 sec. / Average electron dose: 41.34 e/Å2
Details: Each micrograph was acquired as dose-fractionated movies consisting of 62 frames per movie.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 666417
CTF correctionSoftware - Name: cryoSPARC (ver. 2)
Details: Particles were CTF-corrected during projection matching and back projection
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 2)
Details: A three-class ab-initio reconstruction was used for the final 3D classification.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 139296
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

3p8c

,

3sbd

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7use:
Cryo-EM structure of WAVE regulatory complex with Rac1 bound on both A and D site

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