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- EMDB-29023: Structure of Mce1-LucB complex from Mycobacterium smegmatis (Map1) -

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Basic information

Entry
Database: EMDB / ID: EMD-29023
TitleStructure of Mce1-LucB complex from Mycobacterium smegmatis (Map1)
Map dataComposite density map (Map1) for particles in Class1. Map was generated by in PHENIX by using the raw map (boxsize 640 px) and combining Map1a,1b,1c,1d (boxsize 360 px).
Sample
  • Complex: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG) bound to accessory factor LucB
    • Protein or peptide: x 10 types
  • Ligand: x 1 types
KeywordsMembrane protein complex / ABC transporter / Virulence factor / Lipid transport / MEMBRANE PROTEIN
Function / homology
Function and homology information


ATP-binding cassette (ABC) transporter complex / bioluminescence / generation of precursor metabolites and energy / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
Virulence factor Mce protein / Mammalian cell entry, C-terminal / Cholesterol uptake porter CUP1 of Mce4, putative / ABC transporter permease MalE / Permease MlaE / Mce/MlaD / MlaD protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein ...Virulence factor Mce protein / Mammalian cell entry, C-terminal / Cholesterol uptake porter CUP1 of Mce4, putative / ABC transporter permease MalE / Permease MlaE / Mce/MlaD / MlaD protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Prokaryotic membrane lipoprotein lipid attachment site profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ABC-transporter integral membrane protein / Virulence factor Mce family protein / Virulence factor Mce family protein / Virulence factor mce family protein / Virulence factor Mce family protein / Mce-family protein mce1f / ABC transporter, ATP-binding protein / Transmembrane protein / Conserved hypothetical integral membrane protein Yrbe1a / MCE-family protein MCE1c / Green fluorescent protein
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.76 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 6, 2022-
Header (metadata) releaseFeb 22, 2023-
Map releaseFeb 22, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29023.png

Downloads & links

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Map

FileDownload / File: emd_29023.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite density map (Map1) for particles in Class1. Map was generated by in PHENIX by using the raw map (boxsize 640 px) and combining Map1a,1b,1c,1d (boxsize 360 px).
Voxel sizeX=Y=Z: 0.8255 Å
Density
Contour LevelBy AUTHOR: 8.0
Minimum - Maximum-41.32132 - 62.917029999999997
Average (Standard dev.)-0.007667814 (±0.9284523)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 528.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Half map A for raw map used as...

Fileemd_29023_additional_1.map
AnnotationHalf map A for raw map used as template to generate composite half map A.
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Additional map: Half map A for Map1c

Fileemd_29023_additional_10.map
AnnotationHalf map A for Map1c
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Additional map: Half map B for Map1c

Fileemd_29023_additional_11.map
AnnotationHalf map B for Map1c
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Additional map: Constituent Map1c used to in composite Map1. Map...

Fileemd_29023_additional_12.map
AnnotationConstituent Map1c used to in composite Map1. Map was generated by locally refining Class1 particles that were re-centered and extracted with a smaller boxsize (360 px).
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Additional map: Half map A for Map1d

Fileemd_29023_additional_13.map
AnnotationHalf map A for Map1d
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Additional map: Half map B for Map1d

Fileemd_29023_additional_14.map
AnnotationHalf map B for Map1d
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Additional map: Constituent Map1d used to in composite Map1. Map...

Fileemd_29023_additional_15.map
AnnotationConstituent Map1d used to in composite Map1. Map was generated by locally refining Class1 particles that were re-centered and extracted with a smaller boxsize (360 px).
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Additional map: Half map B for raw map used as...

Fileemd_29023_additional_2.map
AnnotationHalf map B for raw map used as template to generate composite half map B.
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Additional map: Raw map used as template to generate composite...

Fileemd_29023_additional_3.map
AnnotationRaw map used as template to generate composite Map1. Map was generated by aligning Class1 particles that were re-extracted with a larger boxsize (640 px).
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Additional map: Half map A for Map1a

Fileemd_29023_additional_4.map
AnnotationHalf map A for Map1a
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Additional map: Half map B for Map1a

Fileemd_29023_additional_5.map
AnnotationHalf map B for Map1a
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Additional map: Constituent Map1a used to in composite Map1. Map...

Fileemd_29023_additional_6.map
AnnotationConstituent Map1a used to in composite Map1. Map was generated by locally refining Class1 particles that were re-centered and extracted with a smaller boxsize (360 px).
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Additional map: Half map A for Map1b

Fileemd_29023_additional_7.map
AnnotationHalf map A for Map1b
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Additional map: Half map B for Map1b

Fileemd_29023_additional_8.map
AnnotationHalf map B for Map1b
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Additional map: Constituent Map1b used to in composite Map1. Map...

Fileemd_29023_additional_9.map
AnnotationConstituent Map1b used to in composite Map1. Map was generated by locally refining Class1 particles that were re-centered and extracted with a smaller boxsize (360 px).
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Half map: Composite half map A for Map1

Fileemd_29023_half_map_1.map
AnnotationComposite half map A for Map1
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Half map: Composite half map B for Map1

Fileemd_29023_half_map_2.map
AnnotationComposite half map B for Map1
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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) bound to accessory factor LucB
Components
  • Complex: Mce1 lipid transporter composed of Mce1 MCE proteins (Mce1ABCDEF) and an ABC transporter (YrbE1A-B, 2 copies of MceG) bound to accessory factor LucB
    • Protein or peptide: Virulence factor Mce family protein
    • Protein or peptide: Virulence factor Mce family protein
    • Protein or peptide: MCE-family protein MCE1c
    • Protein or peptide: Virulence factor mce family protein
    • Protein or peptide: Virulence factor Mce family protein
    • Protein or peptide: Mce-family protein mce1f
    • Protein or peptide: ABC transporter, ATP-binding protein,Green fluorescent protein chimeraATP-binding cassette transporter
    • Protein or peptide: Conserved hypothetical integral membrane protein Yrbe1a
    • Protein or peptide: ABC-transporter integral membrane protein
    • Protein or peptide: Transmembrane protein
  • Ligand: UNKNOWN LIGAND

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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) bound to accessory factor LucB
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
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

MacromoleculeName: Virulence factor Mce family protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 43.944492 KDa
SequenceString: MTEPPAPTAP LNKPKTPPYK LAGLILGLVG VLVLALTWMQ FRGQFEDKVQ LTVLSGRAGL SMDPGSKVTF NGVPIGRLAS IDVVEVDDN PEARLTLDVD PKYLDLIPEN ANVELRATTV FGNKYISFLS PKNPSAERLS ASTPIRAQGV TTEFNTLFET I TAISEQVD ...String:
MTEPPAPTAP LNKPKTPPYK LAGLILGLVG VLVLALTWMQ FRGQFEDKVQ LTVLSGRAGL SMDPGSKVTF NGVPIGRLAS IDVVEVDDN PEARLTLDVD PKYLDLIPEN ANVELRATTV FGNKYISFLS PKNPSAERLS ASTPIRAQGV TTEFNTLFET I TAISEQVD PIKLNETLTA AAQALDGLGD KFGRSIVDGN AILADVNPRM PQIRRDITGL ANLGEVYADA SPDLFDGLDN AV TTARTLN EQRGNLDQAL VAAVGFGNTG GDIFERGGPY LVRGAQDLLP TSALLDEYSP ALFCTIRNYH DAAPKLAGAL GGN GYSLLT NSLVVGVGNP YVYPDNLPRV NAKGGPEGRP GCWQPITRDL WPFPYLVMDT GASIAPYNHF ELGQPMFAEY VWGR QVGEN TINP

UniProtKB: Virulence factor Mce family protein

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Macromolecule #2: Virulence factor Mce family protein

MacromoleculeName: Virulence factor Mce family protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 37.467738 KDa
SequenceString: MSIKGTLFKL GIFSLVLLTF TALIFVVFGQ IRFNRTTEYS AIFKNVSGLR DGQFVRAAGV EVGKVKSVDL INGGEQAEVK FTVERSLPL FQETTAAIRY QDLIGNRYLE LKRGDSDQIL PPGSTIPVER TEPALDLDAL VGGFRPLFRS LEPEKVNTIA T SLITIFQG ...String:
MSIKGTLFKL GIFSLVLLTF TALIFVVFGQ IRFNRTTEYS AIFKNVSGLR DGQFVRAAGV EVGKVKSVDL INGGEQAEVK FTVERSLPL FQETTAAIRY QDLIGNRYLE LKRGDSDQIL PPGSTIPVER TEPALDLDAL VGGFRPLFRS LEPEKVNTIA T SLITIFQG QGGTINDILD QTAQLTASLA DRDQAIGEVI KNLNTVLDTT VRHQKQFDET LVNFETLITG LKNRADPIAT SV ADISDAA GSLADLLSDN RPLLKDTIGY LDVIQAPLVE QKQEVSDILV QMPQALKIIG RAGGIYGDFF NFYACDLTLK LNG LQPGGP VRTVRITTQP SGRCTPK

UniProtKB: Virulence factor Mce family protein

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Macromolecule #3: MCE-family protein MCE1c

MacromoleculeName: MCE-family protein MCE1c / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 54.737805 KDa
SequenceString: MRTLQGSDRF RKGLMGVIVV ALIIGVGSTL TSVPMLFAVP TYYGQFADTG GLNIGDKVRI AGMDVGNVKS MEIDGDKVVI GYTLGGRTI GTESRAAIRT DTILGRKNIE IEPRGSETLK PRGVLPVGQT SAPYQIYDAF LDVTRNAAGW DTQAVRQSLN V LSETVDQT ...String:
MRTLQGSDRF RKGLMGVIVV ALIIGVGSTL TSVPMLFAVP TYYGQFADTG GLNIGDKVRI AGMDVGNVKS MEIDGDKVVI GYTLGGRTI GTESRAAIRT DTILGRKNIE IEPRGSETLK PRGVLPVGQT SAPYQIYDAF LDVTRNAAGW DTQAVRQSLN V LSETVDQT SPHLSAALDG VARFSETIGK RDEDVKKLLA SANKVATVLG DRSTQVNQLL VNAQTLLAAV NERGRSVSLL LE RVSSVSR QVEGFVDENP NLNHVLEQLR TVSDVLNERK QDLADILTVA GKFITSLAEA LASGPYFKVM LVNLIPPTIL QPF VDAAFK KRGIDPEEFW RNAGLPAFRF PDPNGERHEN GAPPAAPTPL EGTPEHPGPA VPPGSPCSYT PPADGIPSPG NPLP CAHLS QGPYGPVPGG YPPPNVATSA PNPDGIAHSP GVPSAAIPGQ MPPEQPGAPV EIAPGPPGAR TVPVSPIPGA PDFTP GIAP PPPAITGPPP PPGPGPQLAP VGEAPLPGNP PFLPPGSQSR

UniProtKB: MCE-family protein MCE1c

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Macromolecule #4: Virulence factor mce family protein

MacromoleculeName: Virulence factor mce family protein / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 58.054551 KDa
SequenceString: MSTIFNIRNI QLPRLSRAAV IIGALVVAAA LVAGYFGMNA YRKLTNTTVT AYFPEVLALY PGDKVLIMGV RVGSIDSIET AGDKMKVVF HFNNKYKVPE NATASILNPS LVASRVIQLS PPYTGGPTLR DGAVLDVDRT QVPIEYDEVR NQVTRLLADL G PTPEQPKG ...String:
MSTIFNIRNI QLPRLSRAAV IIGALVVAAA LVAGYFGMNA YRKLTNTTVT AYFPEVLALY PGDKVLIMGV RVGSIDSIET AGDKMKVVF HFNNKYKVPE NATASILNPS LVASRVIQLS PPYTGGPTLR DGAVLDVDRT QVPIEYDEVR NQVTRLLADL G PTPEQPKG PFGDIIESFA DGFAGKGEQL NRTLRGLSDA LTALNEGRGD FFAVVKSLAL FVNALHRSDQ QFVALNNDLA QF TNSFTNT DQELANALQD LNRVLKTTRE FLDRNGGVLT HDIDNLEQVT TAILQPEPRD GLETGLHAYP NLAANVLNIN SPN QGGIIG LPVLPGVTNF SNPLQFVCSS IQAGSRLGYQ ESAELCAQYL APIMDAIKFN YLPFGMNLAS TAMTLPKQIA YSEK RLQPP PGYKDTTVPG IWSRDTLFSH GNHEPGWIVA PGMQGVQVQP ATANMLTPES LAELLGGPDI VPPPAPPAFG TTRGG NLPG PPNAFDENNP LPPPWYPQPG PPPAPAPGVI PGDPLSAVAP AAPAAPAAPA PAGPPLPAEA GAG

UniProtKB: Virulence factor mce family protein

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Macromolecule #5: Virulence factor Mce family protein

MacromoleculeName: Virulence factor Mce family protein / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 42.566445 KDa
SequenceString: MRLLKGFPKM RNWTRVGRRT AVLAAVALVL TSCGQWRGIA NVPLPGGPGT ESGSMTLYVQ MPETLALNAN SRVRVRDVFV GRVRKIELI NWVPTLTVDV EPGIKLPKNT LAKIGQTSLL GSQHVELNPP EDPSSELLRD GDTIPLAQSS AYPTIERTLA G ISGILTGG ...String:
MRLLKGFPKM RNWTRVGRRT AVLAAVALVL TSCGQWRGIA NVPLPGGPGT ESGSMTLYVQ MPETLALNAN SRVRVRDVFV GRVRKIELI NWVPTLTVDV EPGIKLPKNT LAKIGQTSLL GSQHVELNPP EDPSSELLRD GDTIPLAQSS AYPTIERTLA G ISGILTGG GIPNIEVIQT EVFNILNGRA DQIREFLNQL DTFTDELNQQ REEITRAIDS TNRLLNIVSQ RNDTLDRVLT EF PPLIQHF AETRDLFADA VTALGRLSAA ADETLSGSNA NLHTNLQNLQ RPLKQLGRAA PYLVGALKLI LTVPFNIDNI PKA IRGDYI NVSLKLDLTL SSVDNAFLSG TGVSGMLRAL EQAWGRDPAT MIPDVRFTPN PHDAPGGPLV ERGE

UniProtKB: Virulence factor Mce family protein

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Macromolecule #6: Mce-family protein mce1f

MacromoleculeName: Mce-family protein mce1f / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 54.342898 KDa
SequenceString: MLLTRFIKMQ LVIFLTLTLV ALVVLALFYL RLPTWAGLGM YKLNADLPNS GGLYATANVT YRGTTIGKVT SVEPSESGAR VEMNIYDRY KIPADATANV HSVSAVGEQF IDLTSDSGGG AYFQPGDTIT KATVPAEVGP ALDAAEKGLA VLPKEKIGTL L DEAATAFG ...String:
MLLTRFIKMQ LVIFLTLTLV ALVVLALFYL RLPTWAGLGM YKLNADLPNS GGLYATANVT YRGTTIGKVT SVEPSESGAR VEMNIYDRY KIPADATANV HSVSAVGEQF IDLTSDSGGG AYFQPGDTIT KATVPAEVGP ALDAAEKGLA VLPKEKIGTL L DEAATAFG GLGPSLQRLV DSTQAIAGDF RANIDPVNDI IENSGPIIDS QVNSGDAIQR WAANLNTLAA QSAQNDEALR SG LQQAAPT ADQLNAVFSD VRESLPQTLA NLEIVIDMLK RYNKNVEQVL VALPQGAAVA QTGTIFAPEG LLHFGLGINA PPP CLTGFL PASQWRSPAD TRTEPLPSGL YCKIPKDAPN AVRGARNYPC ADVPGKRAAT PRECRSDEPY QPLGTNPWYG DPDQ IRNCP APGARCDQPV DPGRVIPAPS INNGLNPLPA SQLPPPEVSS GPSSDPLTAP RGGTVTCSGQ QPNPCIYTPA AGATA TYNP ASGEVVGPGG VKYSVTNSNT PGDDGWKEML APAS

UniProtKB: Mce-family protein mce1f

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Macromolecule #7: ABC transporter, ATP-binding protein,Green fluorescent protein chimera

MacromoleculeName: ABC transporter, ATP-binding protein,Green fluorescent protein chimera
type: protein_or_peptide / ID: 7
Details: C-terminus of MceG is tagged (3C-eGFP-4xGly-Tev-Flag-His6)
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 71.62482 KDa
SequenceString: MGVQIDVTGL SKSFGSSKIW EDVTMSIPAG EVSVLLGPSG TGKSVFLKSL IGLLRPERGS IVIDGTDILQ CSAKELYEIR TLFGVLFQD GALFGSMNIY DNTAFPLREH TKKSESEIRK IVMEKLDLVG MPNDGHKFPG EISGGMRKRA GLARALVLDP E IILCDEPD ...String:
MGVQIDVTGL SKSFGSSKIW EDVTMSIPAG EVSVLLGPSG TGKSVFLKSL IGLLRPERGS IVIDGTDILQ CSAKELYEIR TLFGVLFQD GALFGSMNIY DNTAFPLREH TKKSESEIRK IVMEKLDLVG MPNDGHKFPG EISGGMRKRA GLARALVLDP E IILCDEPD SGLDPVRTAY LSQLLIDINA QIDATVLIVT HNINIARTVP DNMGMLFRKQ LVMFGPREVL LTSEEPVVKQ FL NGRRIGP IGMSEEKDEA TAAAEQALVD AGQHDGGVEE IEGVPPQLQA TPGMPERKAV ARRKARVREI LHTLPPAAQA AIL EELDRD QPQLSAPTTQ TAATAPVENY DDSPTGVIEV PKQAGLSGQP PRSPSSGSSS NSLEVLFQGP TAAAAVSKGE ELFT GVVPI LVELDGDVNG HKFSVSGEGE GDATYGKLTL KFICTTGKLP VPWPTLVTTL TYGVQCFSRY PDHMKQHDFF KSAMP EGYV QERTIFFKDD GNYKTRAEVK FEGDTLVNRI ELKGIDFKED GNILGHKLEY NYNSHNVYIM ADKQKNGIKV NFKIRH NIE DGSVQLADHY QQNTPIGDGP VLLPDNHYLS TQSALSKDPN EKRDHMVLLE FVTAAGITLG MDELYKGGGG ENLYFQD YK DDDDKHHHHH H

UniProtKB: ABC transporter, ATP-binding protein, Green fluorescent protein

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Macromolecule #8: Conserved hypothetical integral membrane protein Yrbe1a

MacromoleculeName: Conserved hypothetical integral membrane protein Yrbe1a
type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 27.674619 KDa
SequenceString: MTASTDGFVD YLRGQLEKPL ATVGGFFKMS VMTGKALFTR PFQWKEFVLQ SWFLIRVAFL PTLAVSIPLT VLIIFTLNIL LAEFGAADV SGAGAALGAV TQLGPLVTVL VVAGAGSTAI CADLGARTVR EEIDALEVLG IDPIERLVVP RVVASTFVAF M LNGAVITI ...String:
MTASTDGFVD YLRGQLEKPL ATVGGFFKMS VMTGKALFTR PFQWKEFVLQ SWFLIRVAFL PTLAVSIPLT VLIIFTLNIL LAEFGAADV SGAGAALGAV TQLGPLVTVL VVAGAGSTAI CADLGARTVR EEIDALEVLG IDPIERLVVP RVVASTFVAF M LNGAVITI GLVGGFFFGV YIQNVSAGAY VSTLTLLTGF PEVLISVVKA TLFGMIAGLV GCYRGLTVAG GSKGVGTAVN ET LVLCVVA LFAVNVVLTT IGVRFGTGR

UniProtKB: Conserved hypothetical integral membrane protein Yrbe1a

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Macromolecule #9: ABC-transporter integral membrane protein

MacromoleculeName: ABC-transporter integral membrane protein / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 30.809025 KDa
SequenceString: MSTVQVLRSR FPRAFSRSSE IAATPARFLD SMGHVAWFVV QAIVHVPHAF RHYRRESLRL VAEIGMGTGA MAVIGGTVAI IGFVTLSAG SLIAIQGFAS LGNIGVEAFT GFFAALANIR VVAPVVTGQA LAATVGAGAT AELGAMRISE EVDALEVMGI K SISYLVST ...String:
MSTVQVLRSR FPRAFSRSSE IAATPARFLD SMGHVAWFVV QAIVHVPHAF RHYRRESLRL VAEIGMGTGA MAVIGGTVAI IGFVTLSAG SLIAIQGFAS LGNIGVEAFT GFFAALANIR VVAPVVTGQA LAATVGAGAT AELGAMRISE EVDALEVMGI K SISYLVST RIMAGAIVII PLYAMAILLS FMSAQLVTTI FYSQSVGTYE HYFHTFLRVD DVMWSFLEVI IMSVIVMLNH CY FGYFASG GAVGVGEAVG RSMRTSLIAI VLVVLLASLA LYGTDPNFNL TV

UniProtKB: ABC-transporter integral membrane protein

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Macromolecule #10: Transmembrane protein

MacromoleculeName: Transmembrane protein / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 20.477287 KDa
SequenceString:
MSKWLLRGVV FATAMVIVRL LQGALVNASP GNAIWFSTGL LVLYAIGVAV WGVLDGRGDA RSNPDPDRRA DLAMTWLLAG LAAGILSGA VSWFIGLFYK SIYTESLLNE ITTFAAFTAL LTFLVAVAGV TIGRWTIDRK APPVTRTRHG LAADDDRADT D VFAAVSAN GAQEHTDTTQ TTPLENPDQP RQS

UniProtKB: Transmembrane protein

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Macromolecule #11: UNKNOWN LIGAND

MacromoleculeName: UNKNOWN LIGAND / type: ligand / ID: 11 / Number of copies: 31 / Formula: UNL
Chemical component information


ChemComp, No image

ChemComp-UNL:
Unknown ligand

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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-HClTris
5.0 mMMgSO4Magnesium sulfate
150.0 mMNaClSodium chlorideSodium 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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.
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.
Initial 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 a and b were combined to make Map1 (containing density for LucB).
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 144822
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
Output model

PDB-8fed:
Structure of Mce1-LucB complex from Mycobacterium smegmatis (Map1)

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