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- EMDB-50584: Cryo-EM structure of the c-di-GMP-saturated Bcs macrocomplex (Bcs... -

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Basic information

Entry
Database: EMDB / ID: EMD-50584
TitleCryo-EM structure of the c-di-GMP-saturated Bcs macrocomplex (BcsABR2Q2E2F2G3) for cellulose secretion of E. coli (filtered to 6A)
Map dataCryo-EM map of the assembled c-di-GMP-saturated Bcs macrocomplex of the E. coli Bcs cellulose secretion system (filtered to 6A)
Sample
  • Complex: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli
    • Protein or peptide: x 17 types
KeywordsBacterial cellulose secretion / MEMBRANE PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsAnso I / Krasteva PV
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)757507European Union
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system.
Authors: Itxaso Anso / Samira Zouhir / Thibault Géry Sana / Petya Violinova Krasteva /
Abstract: Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include ...Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsERQ regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.
History
DepositionJun 7, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50584.png

Downloads & links

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Map

FileDownload / File: emd_50584.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the assembled c-di-GMP-saturated Bcs macrocomplex of the E. coli Bcs cellulose secretion system (filtered to 6A)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 500 pix.
= 419.5 Å		0.84 Å/pix.
x 500 pix.
= 419.5 Å		0.84 Å/pix.
x 500 pix.
= 419.5 Å

Surface

Projections

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Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.839 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.125
Minimum - Maximum-0.26510385 - 0.7286734
Average (Standard dev.)-0.00044671784 (±0.02919585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 419.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EM half-map of the assembled c-di-GMP-saturated Bcs macrocomplex...

Fileemd_50584_half_map_1.map
AnnotationEM half-map of the assembled c-di-GMP-saturated Bcs macrocomplex of the E. coli Bcs cellulose secretion system (filtered to 6A)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half-map of the assembled c-di-GMP-saturated Bcs macrocomplex...

Fileemd_50584_half_map_2.map
AnnotationEM half-map of the assembled c-di-GMP-saturated Bcs macrocomplex of the E. coli Bcs cellulose secretion system (filtered to 6A)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Purified c-di-GMP-saturated Bcs macrocomplex from E. coli

EntireName: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli
Components
  • Complex: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli
    • Protein or peptide: BcsA cellulose synthase
    • Protein or peptide: BcsB cellulose co-polymerase
    • Protein or peptide: BcsR essential cellulose secretion regulator
    • Protein or peptide: BcsR essential cellulose secretion regulator
    • Protein or peptide: BcsQ essential secretion regulator
    • Protein or peptide: BcsQ essential secretion regulator
    • Protein or peptide: BcsB cellulose co-polymerase
    • Protein or peptide: BcsB cellulose co-polymerase
    • Protein or peptide: BcsB cellulose co-polymerase
    • Protein or peptide: BcsB cellulose co-polymerase
    • Protein or peptide: BcsB cellulose co-polymerase
    • Protein or peptide: BcsE cellulose secretion enhancer
    • Protein or peptide: BcsE cellulose secretion enhancer
    • Protein or peptide: BcsF cellulose secretion enhancer
    • Protein or peptide: BcsF cellulose secretion enhancer
    • Protein or peptide: BcsG pEtN-transferase
    • Protein or peptide: BcsG pEtN-transferase

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Supramolecule #1: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli

SupramoleculeName: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Molecular weightTheoretical: 990 KDa

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Macromolecule #1: BcsA cellulose synthase

MacromoleculeName: BcsA cellulose synthase / type: protein_or_peptide / ID: 1
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWLL IGASRKETPK PRRRAFSGLQ NIRGRYHQWM NELPERVSHK TQHLDEKKEL GHLSAGARRL ILGIIVTFSL ILALICVTQP ...String:
MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWLL IGASRKETPK PRRRAFSGLQ NIRGRYHQWM NELPERVSHK TQHLDEKKEL GHLSAGARRL ILGIIVTFSL ILALICVTQP FNPLAQFIFL MLLWGVALIV RRMPGRFSAL MLIVLSLTVS CRYIWWRYTS TLNWDDPVSL VCGLILLFAE TYAWIVLVLG YFQVVWPLNR QPVPLPKDMS LWPSVDIFVP TYNEDLNVVK NTIYASLGID WPKDKLNIWI LDDGGREEFR QFAQNVGVKY IARTTHEHAK AGNINNALKY AKGEFVSIFD CDHVPTRSFL QMTMGWFLKE KQLAMMQTPH HFFSPDPFER NLGRFRKTPN EGTLFYGLVQ DGNDMWDATF FCGSCAVIRR KPLDEIGGIA VETVTEDAHT SLRLHRRGYT SAYMRIPQAA GLATESLSAH IGQRIRWARG MVQIFRLDNP LTGKGLKFAQ RLCYVNAMFH FLSGIPRLIF LTAPLAFLLL HAYIIYAPAL MIALFVLPHM IHASLTNSKI QGKYRHSFWS EIYETVLAWY IAPPTLVALI NPHKGKFNVT AKGGLVEEEY VDWVISRPYI FLVLLNLVGV AVGIWRYFYG PPTEMLTVVV SMVWVFYNLI VLGGAVAVSV ESKQVRRSHR VEMTMPAAIA REDGHLFSCT VQDFSDGGLG IKINGQAQIL EGQKVNLLLK RGQQEYVFPT QVARVMGNEV GLKLMPLTTQ QHIDFVQCTF ARADTWALWQ DSYPEDKPLE SLLDILKLGF RGYRHLAEFA PSSVKGIFRV LTSLVSWVVS FIPRRPERSE TAQPSDQALA QQGSARSSGR TGLEFEEFYP YDVPDYAADY KDDDDKRS

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Macromolecule #2: BcsB cellulose co-polymerase

MacromoleculeName: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 2
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV RLEFVGHYQD VCENPASTTL WLDVGRSSGL DLTYQTLNVK NDLSHFPVPF FDPRDNRTNT LPMVFAGAPD VGLQQASAIV ASWFGSRSGW RGQNFPVLYN QLPDRNAIVF ATNDKRPDFL RDHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGAQTGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTSSGA MAAVIGFQSP YNDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERLWYAL ANHPILLAVL AAISVILLAW VLWRLLRIIS RRRLNPDNE

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Macromolecule #3: BcsR essential cellulose secretion regulator

MacromoleculeName: BcsR essential cellulose secretion regulator / type: protein_or_peptide / ID: 3
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSSHHHHHH HHAAGSNNNE PDTLPDPAIG YIFQNDIVAL KQAFSLPDID YADISQREQL AAALKRWPLL AEFAQQK

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Macromolecule #4: BcsR essential cellulose secretion regulator

MacromoleculeName: BcsR essential cellulose secretion regulator / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSSHHHHHH HHAAGSNNNE PDTLPDPAIG YIFQNDIVAL KQAFSLPDID YADISQREQL AAALKRWPLL AEFAQQK

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Macromolecule #5: BcsQ essential secretion regulator

MacromoleculeName: BcsQ essential secretion regulator / type: protein_or_peptide / ID: 5
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: DEXTRO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI ...String:
MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI NNFRIGSQVQ DDIYQLWLQS QRRLLPMLIH RDEAMAECLA AKQPVGEYRS DALAAEEILT LANWCLLNYS GLKTPVGSKS

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Macromolecule #6: BcsQ essential secretion regulator

MacromoleculeName: BcsQ essential secretion regulator / type: protein_or_peptide / ID: 6 / Enantiomer: DEXTRO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI ...String:
MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSIE EQENPQHWQT RLSDICSGLQ QLKASGRYQW ILIDLPRDAS QITHQLLSLC DHSLAIVNVD ANCHIRLHQQ ALPDGAHILI NNFRIGSQVQ DDIYQLWLQS QRRLLPMLIH RDEAMAECLA AKQPVGEYRS DALAAEEILT LANWCLLNYS GLKTPVGSKS

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Macromolecule #7: BcsB cellulose co-polymerase

MacromoleculeName: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV RLEFVGHYQD VCENPASTTL WLDVGRSSGL DLTYQTLNVK NDLSHFPVPF FDPRDNRTNT LPMVFAGAPD VGLQQASAIV ASWFGSRSGW RGQNFPVLYN QLPDRNAIVF ATNDKRPDFL RDHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGAQTGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTSSGA MAAVIGFQSP YNDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERLWYAL ANHPILLAVL AAISVILLAW VLWRLLRIIS RRRLNPDNE

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Macromolecule #8: BcsB cellulose co-polymerase

MacromoleculeName: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV RLEFVGHYQD VCENPASTTL WLDVGRSSGL DLTYQTLNVK NDLSHFPVPF FDPRDNRTNT LPMVFAGAPD VGLQQASAIV ASWFGSRSGW RGQNFPVLYN QLPDRNAIVF ATNDKRPDFL RDHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGAQTGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTSSGA MAAVIGFQSP YNDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERLWYAL ANHPILLAVL AAISVILLAW VLWRLLRIIS RRRLNPDNE

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Macromolecule #9: BcsB cellulose co-polymerase

MacromoleculeName: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV RLEFVGHYQD VCENPASTTL WLDVGRSSGL DLTYQTLNVK NDLSHFPVPF FDPRDNRTNT LPMVFAGAPD VGLQQASAIV ASWFGSRSGW RGQNFPVLYN QLPDRNAIVF ATNDKRPDFL RDHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGAQTGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTSSGA MAAVIGFQSP YNDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERLWYAL ANHPILLAVL AAISVILLAW VLWRLLRIIS RRRLNPDNE

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Macromolecule #10: BcsB cellulose co-polymerase

MacromoleculeName: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 10 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV RLEFVGHYQD VCENPASTTL WLDVGRSSGL DLTYQTLNVK NDLSHFPVPF FDPRDNRTNT LPMVFAGAPD VGLQQASAIV ASWFGSRSGW RGQNFPVLYN QLPDRNAIVF ATNDKRPDFL RDHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGAQTGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTSSGA MAAVIGFQSP YNDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERLWYAL ANHPILLAVL AAISVILLAW VLWRLLRIIS RRRLNPDNE

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Macromolecule #11: BcsB cellulose co-polymerase

MacromoleculeName: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 11 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVLR GINPNGSIEF GMRSDEVVTK AMLNLEYTPS PSLLPVQSQL KVYLNDELMG VLPVTKEQLG KKTLAQMPIN PLFITDFNRV RLEFVGHYQD VCENPASTTL WLDVGRSSGL DLTYQTLNVK NDLSHFPVPF FDPRDNRTNT LPMVFAGAPD VGLQQASAIV ASWFGSRSGW RGQNFPVLYN QLPDRNAIVF ATNDKRPDFL RDHPAVKAPV IEMINHPQNP YVKLLVVFGR DDKDLLQAAK GIAQGNILFR GESVVVNEVK PLLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQPVQNHV VIGDDSTIDF SKYYHFIPMP DLRAFANAGF PFSRMADLSQ TITVMPKAPN EAQMETLLNT VGFIGAQTGF PAINLTVTDD GSTIQGKDAD IMIIGGIPDK LKDDKQIDLL VQATESWVKT PMRQTPFPGI VPDESDRAAE TRSTLTSSGA MAAVIGFQSP YNDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERLWYAL ANHPILLAVL AAISVILLAW VLWRLLRIIS RRRLNPDNE

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Macromolecule #12: BcsE cellulose secretion enhancer

MacromoleculeName: BcsE cellulose secretion enhancer / type: protein_or_peptide / ID: 12
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASWSHPQFE KGSMRDIVDP VFSIGISSLW DELRHMPAGG VWWFNVDRHE DAISLANQTI ASQAETAHVA VISMDSDPAK IFQLDDSQGP EKIKLFSMLN HEKGLYYLTR DLQCSIDPHN YLFILVCANN AWQNIPAERL RSWLDKMNKW SRLNHCSLLV INPGNNNDKQ ...String:
MASWSHPQFE KGSMRDIVDP VFSIGISSLW DELRHMPAGG VWWFNVDRHE DAISLANQTI ASQAETAHVA VISMDSDPAK IFQLDDSQGP EKIKLFSMLN HEKGLYYLTR DLQCSIDPHN YLFILVCANN AWQNIPAERL RSWLDKMNKW SRLNHCSLLV INPGNNNDKQ FSLLLEEYRS LFGLASLRFQ GDQHLLDIAF WCNEKGVSAR QQLSVQQQNG IWTLVQSEEA EIQPRSDEKR ILSNVAVLEG APPLSEHWQL FNNNEVLFNE ARTAQAATVV FSLQQNAQIE PLARSIHTLR RQRGSAMKIL VRENTASLRA TDERLLLACG ANMVIPWNAP LSRCLTMIES VQGQKFSRYV PEDITTLLSM TQPLKLRGFQ KWDVFCNAVN NMMNNPLLPA HGKGVLVALR PVPGIRVEQA LTLCRPNRTG DIMTIGGNRL VLFLSFCRIN DLDTALNHIF PLPTGDIFSN RMVWFEDDQI SAELVQMRLL APEQWGMPLP LTQSSKPVIN AEHDGRHWRR IPEPMRLLDD AVERSS

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Macromolecule #13: BcsE cellulose secretion enhancer

MacromoleculeName: BcsE cellulose secretion enhancer / type: protein_or_peptide / ID: 13 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASWSHPQFE KGSMRDIVDP VFSIGISSLW DELRHMPAGG VWWFNVDRHE DAISLANQTI ASQAETAHVA VISMDSDPAK IFQLDDSQGP EKIKLFSMLN HEKGLYYLTR DLQCSIDPHN YLFILVCANN AWQNIPAERL RSWLDKMNKW SRLNHCSLLV INPGNNNDKQ ...String:
MASWSHPQFE KGSMRDIVDP VFSIGISSLW DELRHMPAGG VWWFNVDRHE DAISLANQTI ASQAETAHVA VISMDSDPAK IFQLDDSQGP EKIKLFSMLN HEKGLYYLTR DLQCSIDPHN YLFILVCANN AWQNIPAERL RSWLDKMNKW SRLNHCSLLV INPGNNNDKQ FSLLLEEYRS LFGLASLRFQ GDQHLLDIAF WCNEKGVSAR QQLSVQQQNG IWTLVQSEEA EIQPRSDEKR ILSNVAVLEG APPLSEHWQL FNNNEVLFNE ARTAQAATVV FSLQQNAQIE PLARSIHTLR RQRGSAMKIL VRENTASLRA TDERLLLACG ANMVIPWNAP LSRCLTMIES VQGQKFSRYV PEDITTLLSM TQPLKLRGFQ KWDVFCNAVN NMMNNPLLPA HGKGVLVALR PVPGIRVEQA LTLCRPNRTG DIMTIGGNRL VLFLSFCRIN DLDTALNHIF PLPTGDIFSN RMVWFEDDQI SAELVQMRLL APEQWGMPLP LTQSSKPVIN AEHDGRHWRR IPEPMRLLDD AVERSS

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Macromolecule #14: BcsF cellulose secretion enhancer

MacromoleculeName: BcsF cellulose secretion enhancer / type: protein_or_peptide / ID: 14
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MMTISDIIEI IVVCALIFFP LGYLARHSLR RIRDTLRLFF AKPRYVKPAG TLRRTEKARA TKK

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Macromolecule #15: BcsF cellulose secretion enhancer

MacromoleculeName: BcsF cellulose secretion enhancer / type: protein_or_peptide / ID: 15 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MMTISDIIEI IVVCALIFFP LGYLARHSLR RIRDTLRLFF AKPRYVKPAG TLRRTEKARA TKK

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Macromolecule #16: BcsG pEtN-transferase

MacromoleculeName: BcsG pEtN-transferase / type: protein_or_peptide / ID: 16
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPES IMSQGSQVAG FSTDYLIDLV TRFINWQMIG AIFVLLVAWL FLSQWIRITV FVVAILLWLN VLTLAGPSFS LWPAGQPTTT ...String:
MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPES IMSQGSQVAG FSTDYLIDLV TRFINWQMIG AIFVLLVAWL FLSQWIRITV FVVAILLWLN VLTLAGPSFS LWPAGQPTTT VTTTGGNAAA TVAATGGAPV VGDMPAQTAP PTTANLNAWL NNFYNAEAKR KSTFPSSLPA DAQPFELLVI NICSLSWSDI EAAGLMSHPL WSHFDIEFKN FNSATSYSGP AAIRLLRASC GQTSHTNLYQ PANNDCYLFD NLSKLGFTQH LMMGHNGQFG GFLKEVRENG GMQSELMDQT NLPVILLGFD GSPVYDDTAV LNRWLDVTEK DKNSRSATFY NTLPLHDGNH YPGVSKTADY KARAQKFFDE LDAFFTELEK SGRKVMVVVV PEHGGALKGD RMQVSGLRDI PSPSITDVPV GVKFFGMKAP HQGAPIVIEQ PSSFLAISDL VVRVLDGKIF TEDNVDWKKL TSGLHKQHRS PRTQMQ

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Macromolecule #17: BcsG pEtN-transferase

MacromoleculeName: BcsG pEtN-transferase / type: protein_or_peptide / ID: 17 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPES IMSQGSQVAG FSTDYLIDLV TRFINWQMIG AIFVLLVAWL FLSQWIRITV FVVAILLWLN VLTLAGPSFS LWPAGQPTTT ...String:
MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPES IMSQGSQVAG FSTDYLIDLV TRFINWQMIG AIFVLLVAWL FLSQWIRITV FVVAILLWLN VLTLAGPSFS LWPAGQPTTT VTTTGGNAAA TVAATGGAPV VGDMPAQTAP PTTANLNAWL NNFYNAEAKR KSTFPSSLPA DAQPFELLVI NICSLSWSDI EAAGLMSHPL WSHFDIEFKN FNSATSYSGP AAIRLLRASC GQTSHTNLYQ PANNDCYLFD NLSKLGFTQH LMMGHNGQFG GFLKEVRENG GMQSELMDQT NLPVILLGFD GSPVYDDTAV LNRWLDVTEK DKNSRSATFY NTLPLHDGNH YPGVSKTADY KARAQKFFDE LDAFFTELEK SGRKVMVVVV PEHGGALKGD RMQVSGLRDI PSPSITDVPV GVKFFGMKAP HQGAPIVIEQ PSSFLAISDL VVRVLDGKIF TEDNVDWKKL TSGLHKQHRS PRTQMQ

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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

Concentration2 mg/mL
BufferpH: 8
Details: 120 mM NaCL 20 mM HEPES pH8 5 mM MgCl2 10 uM ApppCp 4 uM c-di-GMP 0.01% LM-NPG
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsPurification from solubilized membrane fraction using an anti-FLAG M2 affinity resin

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 20022 / Average electron dose: 49.35 e/Å2
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: 2.1 µm / Nominal defocus min: 0.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1359795
Startup modelType of model: OTHER / Details: Ab Initio cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: OTHER / Software - Name: cryoSPARC
Details: Low-pass filtered for visualization of flexible/lower-resoution features
Number images used: 357003
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model / Details: emd-11836

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