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- EMDB-29239: Local refinement of YrbE and MCE ring in Msmeg Mce1 transporter i... -

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Basic information

Entry
Database: EMDB / ID: EMD-29239
TitleLocal refinement of YrbE and MCE ring in Msmeg Mce1 transporter in the absence of LucB (Map2b)
Map dataConstituent Map2b used to in composite Map2. Map was generated by locally refining Class2 particles that were re-centered and extracted with a smaller boxsize (360 px).
Sample
  • Complex: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG)
    • Protein or peptide: Virulence factor Mce family protein Mce1A
    • Protein or peptide: Virulence factor Mce family protein Mce1B
    • Protein or peptide: Virulence factor Mce family protein Mce1C
    • Protein or peptide: Virulence factor Mce family protein Mce1D
    • Protein or peptide: Virulence factor Mce family protein Mce1E
    • Protein or peptide: Virulence factor Mce family protein Mce1F
    • Protein or peptide: ATP-binding protein MceG
    • Protein or peptide: ATP-binding protein MceG
    • Protein or peptide: ABC transporter permease protein YrbE1A
    • Protein or peptide: ABC transporter permease protein YrbE1B
KeywordsMembrane protein complex / ABC transporter / Virulence factor / Lipid transport / MEMBRANE PROTEIN
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsChen J / Bhabha G / Ekiert DC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)PEW-00033055 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Nature / Year: 2023
Title: Structure of an endogenous mycobacterial MCE lipid transporter.
Authors: James Chen / Alice Fruhauf / Catherine Fan / Jackeline Ponce / Beatrix Ueberheide / Gira Bhabha / Damian C Ekiert /
Abstract: To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host. The mammalian cell entry (MCE) proteins are important virulence ...To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids and cholesterol across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
History
DepositionDec 19, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29239.png

Downloads & links

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Map

FileDownload / File: emd_29239.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConstituent Map2b used to in composite Map2. Map was generated by locally refining Class2 particles that were re-centered and extracted with a smaller boxsize (360 px).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 297.18 Å		0.83 Å/pix.
x 360 pix.
= 297.18 Å		0.83 Å/pix.
x 360 pix.
= 297.18 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8255 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.73770493 - 1.1816484
Average (Standard dev.)0.000934531 (±0.027350478)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.18 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A for Map2b

Fileemd_29239_half_map_1.map
AnnotationHalf map A for Map2b
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B for Map2b

Fileemd_29239_half_map_2.map
AnnotationHalf map B for Map2b
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF)...

EntireName: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG)
Components
  • Complex: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG)
    • Protein or peptide: Virulence factor Mce family protein Mce1A
    • Protein or peptide: Virulence factor Mce family protein Mce1B
    • Protein or peptide: Virulence factor Mce family protein Mce1C
    • Protein or peptide: Virulence factor Mce family protein Mce1D
    • Protein or peptide: Virulence factor Mce family protein Mce1E
    • Protein or peptide: Virulence factor Mce family protein Mce1F
    • Protein or peptide: ATP-binding protein MceG
    • Protein or peptide: ATP-binding protein MceG
    • Protein or peptide: ABC transporter permease protein YrbE1A
    • Protein or peptide: ABC transporter permease protein YrbE1B

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Supramolecule #1: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF)...

SupramoleculeName: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Complex was isolated directly from Mycobacterium smegmatis by pulling down MceG-GFP using GFP-affinity purification and size exclusion chromatography
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Macromolecule #1: Virulence factor Mce family protein Mce1A

MacromoleculeName: Virulence factor Mce family protein Mce1A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTEPPAPTAP LNKPKTPPYK LAGLILGLVG VLVLALTWMQ FRGQFEDKVQ LTVLSGRAGL SMDPGSKVTF NGVPIGRLAS IDVVEVDDNP EARLTLDVDP KYLDLIPENA NVELRATTVF GNKYISFLSP KNPSAERLSA STPIRAQGVT TEFNTLFETI TAISEQVDPI ...String:
MTEPPAPTAP LNKPKTPPYK LAGLILGLVG VLVLALTWMQ FRGQFEDKVQ LTVLSGRAGL SMDPGSKVTF NGVPIGRLAS IDVVEVDDNP EARLTLDVDP KYLDLIPENA NVELRATTVF GNKYISFLSP KNPSAERLSA STPIRAQGVT TEFNTLFETI TAISEQVDPI KLNETLTAAA QALDGLGDKF GRSIVDGNAI LADVNPRMPQ IRRDITGLAN LGEVYADASP DLFDGLDNAV TTARTLNEQR GNLDQALVAA VGFGNTGGDI FERGGPYLVR GAQDLLPTSA LLDEYSPALF CTIRNYHDAA PKLAGALGGN GYSLLTNSLV VGVGNPYVYP DNLPRVNAKG GPEGRPGCWQ PITRDLWPFP YLVMDTGASI APYNHFELGQ PMFAEYVWGR QVGENTINP

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Macromolecule #2: Virulence factor Mce family protein Mce1B

MacromoleculeName: Virulence factor Mce family protein Mce1B / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSIKGTLFKL GIFSLVLLTF TALIFVVFGQ IRFNRTTEYS AIFKNVSGLR DGQFVRAAGV EVGKVKSVDL INGGEQAEVK FTVERSLPLF QETTAAIRYQ DLIGNRYLEL KRGDSDQILP PGSTIPVERT EPALDLDALV GGFRPLFRSL EPEKVNTIAT SLITIFQGQG ...String:
MSIKGTLFKL GIFSLVLLTF TALIFVVFGQ IRFNRTTEYS AIFKNVSGLR DGQFVRAAGV EVGKVKSVDL INGGEQAEVK FTVERSLPLF QETTAAIRYQ DLIGNRYLEL KRGDSDQILP PGSTIPVERT EPALDLDALV GGFRPLFRSL EPEKVNTIAT SLITIFQGQG GTINDILDQT AQLTASLADR DQAIGEVIKN LNTVLDTTVR HQKQFDETLV NFETLITGLK NRADPIATSV ADISDAAGSL ADLLSDNRPL LKDTIGYLDV IQAPLVEQKQ EVSDILVQMP QALKIIGRAG GIYGDFFNFY ACDLTLKLNG LQPGGPVRTV RITTQPSGRC TPK

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Macromolecule #3: Virulence factor Mce family protein Mce1C

MacromoleculeName: Virulence factor Mce family protein Mce1C / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MRTLQGSDRF RKGLMGVIVV ALIIGVGSTL TSVPMLFAVP TYYGQFADTG GLNIGDKVRI AGMDVGNVKS MEIDGDKVVI GYTLGGRTIG TESRAAIRTD TILGRKNIEI EPRGSETLKP RGVLPVGQTS APYQIYDAFL DVTRNAAGWD TQAVRQSLNV LSETVDQTSP ...String:
MRTLQGSDRF RKGLMGVIVV ALIIGVGSTL TSVPMLFAVP TYYGQFADTG GLNIGDKVRI AGMDVGNVKS MEIDGDKVVI GYTLGGRTIG TESRAAIRTD TILGRKNIEI EPRGSETLKP RGVLPVGQTS APYQIYDAFL DVTRNAAGWD TQAVRQSLNV LSETVDQTSP HLSAALDGVA RFSETIGKRD EDVKKLLASA NKVATVLGDR STQVNQLLVN AQTLLAAVNE RGRSVSLLLE RVSSVSRQVE GFVDENPNLN HVLEQLRTVS DVLNERKQDL ADILTVAGKF ITSLAEALAS GPYFKVMLVN LIPPTILQPF VDAAFKKRGI DPEEFWRNAG LPAFRFPDPN GERHENGAPP AAPTPLEGTP EHPGPAVPPG SPCSYTPPAD GIPSPGNPLP CAHLSQGPYG PVPGGYPPPN VATSAPNPDG IAHSPGVPSA AIPGQMPPEQ PGAPVEIAPG PPGARTVPVS PIPGAPDFTP GIAPPPPAIT GPPPPPGPGP QLAPVGEAPL PGNPPFLPPG SQSR

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Macromolecule #4: Virulence factor Mce family protein Mce1D

MacromoleculeName: Virulence factor Mce family protein Mce1D / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSTIFNIRNI QLPRLSRAAV IIGALVVAAA LVAGYFGMNA YRKLTNTTVT AYFPEVLALY PGDKVLIMGV RVGSIDSIET AGDKMKVVFH FNNKYKVPEN ATASILNPSL VASRVIQLSP PYTGGPTLRD GAVLDVDRTQ VPIEYDEVRN QVTRLLADLG PTPEQPKGPF ...String:
MSTIFNIRNI QLPRLSRAAV IIGALVVAAA LVAGYFGMNA YRKLTNTTVT AYFPEVLALY PGDKVLIMGV RVGSIDSIET AGDKMKVVFH FNNKYKVPEN ATASILNPSL VASRVIQLSP PYTGGPTLRD GAVLDVDRTQ VPIEYDEVRN QVTRLLADLG PTPEQPKGPF GDIIESFADG FAGKGEQLNR TLRGLSDALT ALNEGRGDFF AVVKSLALFV NALHRSDQQF VALNNDLAQF TNSFTNTDQE LANALQDLNR VLKTTREFLD RNGGVLTHDI DNLEQVTTAI LQPEPRDGLE TGLHAYPNLA ANVLNINSPN QGGIIGLPVL PGVTNFSNPL QFVCSSIQAG SRLGYQESAE LCAQYLAPIM DAIKFNYLPF GMNLASTAMT LPKQIAYSEK RLQPPPGYKD TTVPGIWSRD TLFSHGNHEP GWIVAPGMQG VQVQPATANM LTPESLAELL GGPDIVPPPA PPAFGTTRGG NLPGPPNAFD ENNPLPPPWY PQPGPPPAPA PGVIPGDPLS AVAPAAPAAP AAPAPAGPPL PAEAGAG

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Macromolecule #5: Virulence factor Mce family protein Mce1E

MacromoleculeName: Virulence factor Mce family protein Mce1E / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MRLLKGFPKM RNWTRVGRRT AVLAAVALVL TSCGQWRGIA NVPLPGGPGT ESGSMTLYVQ MPETLALNAN SRVRVRDVFV GRVRKIELIN WVPTLTVDVE PGIKLPKNTL AKIGQTSLLG SQHVELNPPE DPSSELLRDG DTIPLAQSSA YPTIERTLAG ISGILTGGGI ...String:
MRLLKGFPKM RNWTRVGRRT AVLAAVALVL TSCGQWRGIA NVPLPGGPGT ESGSMTLYVQ MPETLALNAN SRVRVRDVFV GRVRKIELIN WVPTLTVDVE PGIKLPKNTL AKIGQTSLLG SQHVELNPPE DPSSELLRDG DTIPLAQSSA YPTIERTLAG ISGILTGGGI PNIEVIQTEV FNILNGRADQ IREFLNQLDT FTDELNQQRE EITRAIDSTN RLLNIVSQRN DTLDRVLTEF PPLIQHFAET RDLFADAVTA LGRLSAAADE TLSGSNANLH TNLQNLQRPL KQLGRAAPYL VGALKLILTV PFNIDNIPKA IRGDYINVSL KLDLTLSSVD NAFLSGTGVS GMLRALEQAW GRDPATMIPD VRFTPNPHDA PGGPLVERGE

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Macromolecule #6: Virulence factor Mce family protein Mce1F

MacromoleculeName: Virulence factor Mce family protein Mce1F / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MLLTRFIKMQ LVIFLTLTLV ALVVLALFYL RLPTWAGLGM YKLNADLPNS GGLYATANVT YRGTTIGKVT SVEPSESGAR VEMNIYDRYK IPADATANVH SVSAVGEQFI DLTSDSGGGA YFQPGDTITK ATVPAEVGPA LDAAEKGLAV LPKEKIGTLL DEAATAFGGL ...String:
MLLTRFIKMQ LVIFLTLTLV ALVVLALFYL RLPTWAGLGM YKLNADLPNS GGLYATANVT YRGTTIGKVT SVEPSESGAR VEMNIYDRYK IPADATANVH SVSAVGEQFI DLTSDSGGGA YFQPGDTITK ATVPAEVGPA LDAAEKGLAV LPKEKIGTLL DEAATAFGGL GPSLQRLVDS TQAIAGDFRA NIDPVNDIIE NSGPIIDSQV NSGDAIQRWA ANLNTLAAQS AQNDEALRSG LQQAAPTADQ LNAVFSDVRE SLPQTLANLE IVIDMLKRYN KNVEQVLVAL PQGAAVAQTG TIFAPEGLLH FGLGINAPPP CLTGFLPASQ WRSPADTRTE PLPSGLYCKI PKDAPNAVRG ARNYPCADVP GKRAATPREC RSDEPYQPLG TNPWYGDPDQ IRNCPAPGAR CDQPVDPGRV IPAPSINNGL NPLPASQLPP PEVSSGPSSD PLTAPRGGTV TCSGQQPNPC IYTPAAGATA TYNPASGEVV GPGGVKYSVT NSNTPGDDGW KEMLAPAS

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Macromolecule #7: ATP-binding protein MceG

MacromoleculeName: ATP-binding protein MceG / type: protein_or_peptide / ID: 7
Details: C-terminus of MceG is tagged (3C-eGFP-4xGly-Tev-Flag-His6).
Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MGVQIDVTGL SKSFGSSKIW EDVTMSIPAG EVSVLLGPSG TGKSVFLKSL IGLLRPERGS IVIDGTDILQ CSAKELYEIR TLFGVLFQDG ALFGSMNIYD NTAFPLREHT KKSESEIRKI VMEKLDLVGM PNDGHKFPGE ISGGMRKRAG LARALVLDPE IILCDEPDSG ...String:
MGVQIDVTGL SKSFGSSKIW EDVTMSIPAG EVSVLLGPSG TGKSVFLKSL IGLLRPERGS IVIDGTDILQ CSAKELYEIR TLFGVLFQDG ALFGSMNIYD NTAFPLREHT KKSESEIRKI VMEKLDLVGM PNDGHKFPGE ISGGMRKRAG LARALVLDPE IILCDEPDSG LDPVRTAYLS QLLIDINAQI DATVLIVTHN INIARTVPDN MGMLFRKQLV MFGPREVLLT SEEPVVKQFL NGRRIGPIGM SEEKDEATAA AEQALVDAGQ HDGGVEEIEG VPPQLQATPG MPERKAVARR KARVREILHT LPPAAQAAIL EELDRDQPQL SAPTTQTAAT APVENYDDSP TGVIEVPKQA GLSGQPPRSP SSGSSSNSLE VLFQGPTAAA AVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT LVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN GIKVNFKIRH NIEDGSVQLA DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYKG GGGENLYFQD YKDDDDKHHH HHH

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Macromolecule #8: ATP-binding protein MceG

MacromoleculeName: ATP-binding protein MceG / type: protein_or_peptide / ID: 8
Details: C-terminus of MceG is tagged (3C-eGFP-4xGly-Tev-Flag-His6).
Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MGVQIDVTGL SKSFGSSKIW EDVTMSIPAG EVSVLLGPSG TGKSVFLKSL IGLLRPERGS IVIDGTDILQ CSAKELYEIR TLFGVLFQDG ALFGSMNIYD NTAFPLREHT KKSESEIRKI VMEKLDLVGM PNDGHKFPGE ISGGMRKRAG LARALVLDPE IILCDEPDSG ...String:
MGVQIDVTGL SKSFGSSKIW EDVTMSIPAG EVSVLLGPSG TGKSVFLKSL IGLLRPERGS IVIDGTDILQ CSAKELYEIR TLFGVLFQDG ALFGSMNIYD NTAFPLREHT KKSESEIRKI VMEKLDLVGM PNDGHKFPGE ISGGMRKRAG LARALVLDPE IILCDEPDSG LDPVRTAYLS QLLIDINAQI DATVLIVTHN INIARTVPDN MGMLFRKQLV MFGPREVLLT SEEPVVKQFL NGRRIGPIGM SEEKDEATAA AEQALVDAGQ HDGGVEEIEG VPPQLQATPG MPERKAVARR KARVREILHT LPPAAQAAIL EELDRDQPQL SAPTTQTAAT APVENYDDSP TGVIEVPKQA GLSGQPPRSP SSGSSSNSLE VLFQGPTAAA AVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT LVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN GIKVNFKIRH NIEDGSVQLA DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYKG GGGENLYFQD YKDDDDKHHH HHH

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Macromolecule #9: ABC transporter permease protein YrbE1A

MacromoleculeName: ABC transporter permease protein YrbE1A / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MTASTDGFVD YLRGQLEKPL ATVGGFFKMS VMTGKALFTR PFQWKEFVLQ SWFLIRVAFL PTLAVSIPLT VLIIFTLNIL LAEFGAADVS GAGAALGAVT QLGPLVTVLV VAGAGSTAIC ADLGARTVRE EIDALEVLGI DPIERLVVPR VVASTFVAFM LNGAVITIGL ...String:
MTASTDGFVD YLRGQLEKPL ATVGGFFKMS VMTGKALFTR PFQWKEFVLQ SWFLIRVAFL PTLAVSIPLT VLIIFTLNIL LAEFGAADVS GAGAALGAVT QLGPLVTVLV VAGAGSTAIC ADLGARTVRE EIDALEVLGI DPIERLVVPR VVASTFVAFM LNGAVITIGL VGGFFFGVYI QNVSAGAYVS TLTLLTGFPE VLISVVKATL FGMIAGLVGC YRGLTVAGGS KGVGTAVNET LVLCVVALFA VNVVLTTIGV RFGTGR

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Macromolecule #10: ABC transporter permease protein YrbE1B

MacromoleculeName: ABC transporter permease protein YrbE1B / type: protein_or_peptide / ID: 10 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MSTVQVLRSR FPRAFSRSSE IAATPARFLD SMGHVAWFVV QAIVHVPHAF RHYRRESLRL VAEIGMGTGA MAVIGGTVAI IGFVTLSAGS LIAIQGFASL GNIGVEAFTG FFAALANIRV VAPVVTGQAL AATVGAGATA ELGAMRISEE VDALEVMGIK SISYLVSTRI ...String:
MSTVQVLRSR FPRAFSRSSE IAATPARFLD SMGHVAWFVV QAIVHVPHAF RHYRRESLRL VAEIGMGTGA MAVIGGTVAI IGFVTLSAGS LIAIQGFASL GNIGVEAFTG FFAALANIRV VAPVVTGQAL AATVGAGATA ELGAMRISEE VDALEVMGIK SISYLVSTRI MAGAIVIIPL YAMAILLSFM SAQLVTTIFY SQSVGTYEHY FHTFLRVDDV MWSFLEVIIM SVIVMLNHCY FGYFASGGAV GVGEAVGRSM RTSLIAIVLV VLLASLALYG TDPNFNLTV

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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

Concentration1.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC4H13Cl2NO3Tris-HCl
5.0 mMMgSO4Magnesium sulfate
150.0 mMNaClSodium chloride
1.0 mMC24H46O11n-dodecyl-beta-D-maltoside
1.0 mMC4H10O2S2Dithiothreitol

Details: 50 mM Tris-HCl pH 7.5, 5 mM MgSO4, 150 mM NaCl, 1 mM DDM, 1 mM DTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample contains a mixture of MCE proteins endogenously purified from Mycobacterium smegmatis.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 2 / Number real images: 43925 / Average exposure time: 2.0 sec. / Average electron dose: 60.0 e/Å2
Details: Images were collected in super resolution mode (nominal pixel size of 0.8255 A/pix after binning by 2).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 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: 2869223
Startup modelType of model: OTHER
Details: Starting model was generated using cryoSPARC Ab initio Reconstruction.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 179480
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 85000 / Software - Name: cryoSPARC (ver. 3.3.1)
Details: Consensus set of particles were 3D classified using cryoSPARC Heterogenous Refinement (four classes, a-d). Class c and d were combined to make Map2 (containing no density for LucB).
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel was initial fitted into the map using Chimera follow-by rigid body refinement in PHENIX. Models were further refined using PHENIX real-space refinement and then manually inspected in Coot.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient

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