EMDB-50595: Cryo-EM structure of the c-di-GMP non-saturated Bcs macrocomplex ...

Basic information

Entry

Database: EMDB / ID: EMD-50595

• Title: Cryo-EM structure of the c-di-GMP non-saturated Bcs macrocomplex (BcsABR2Q2E2F2G3) for cellulose secretion of E. coli (filtered to 7A)
• Map data: Cryo-EM structure of the c-di-GMP non-saturated Bcs macrocomplex (BcsABR2Q2E2F2G3) for cellulose secretion of E. coli (filtered to 7A)

Sample

• Complex: Purified c-di-GMP-saturated Bcs macrocomplex from E. coli
  • Protein or peptide: x 18 types

• Keywords: Bacterial cellulose secretion / MEMBRANE PROTEIN
• Biological species: Escherichia coli (E. coli)
• Method: single particle reconstruction / cryo EM / Resolution: 7.0 Å
• Authors: Anso I / Krasteva PV

Funding support

European Union, 1 items

Organization	Grant number	Country
European Research Council (ERC)	757507	European Union

• Citation: Journal: 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 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

Deposition	Jun 10, 2024	-
Header (metadata) release	Oct 16, 2024	-
Map release	Oct 16, 2024	-
Update	Oct 23, 2024	-
Current status	Oct 23, 2024	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_50595.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Cryo-EM structure of the c-di-GMP non-saturated Bcs macrocomplex (BcsABR2Q2E2F2G3) for cellulose secretion of E. coli (filtered to 7A)
• Voxel size: X=Y=Z: 0.839 Å

Density

Contour Level	By AUTHOR: 0.09
Minimum - Maximum	-0.25324005 - 0.69335914
Average (Standard dev.)	-0.00047355925 (±0.027992588)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 500 500 500 Spacing 500 500 500
Cell	A=B=C: 419.5 Å α=β=γ: 90.0 °

Supplemental data

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

• File: emd_50595_half_map_1.map
• Annotation: EM half-map of the c-di-GMP non-saturated Bcs macrocomplex (BcsABR2Q2E2F2G3) for cellulose secretion of E. coli (filtered to 7A)

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

• File: emd_50595_half_map_2.map
• Annotation: EM half-map of the c-di-GMP non-saturated Bcs macrocomplex (BcsABR2Q2E2F2G3) for cellulose secretion of E. coli (filtered to 7A)

Sample components

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

• Entire: Name: 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: 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: BcsR essential cellulose secretion regulator
  • Protein or peptide: BcsR essential cellulose secretion regulator
  • Protein or peptide: BcsQ essential cellulose secretion regulator
  • Protein or peptide: BcsQ essential cellulose secretion regulator
  • Protein or peptide: BcsF cellulose secretion enhancer
  • Protein or peptide: BcsF cellulose secretion enhancer
  • Protein or peptide: BcsE cellulose secretion enhancer
  • Protein or peptide: BcsE cellulose secretion enhancer
  • Protein or peptide: BcsG pEtN-transferase
  • Protein or peptide: BcsG pEtN-transferase
  • Protein or peptide: BcsG pEtN-transferase

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

• Supramolecule: Name: 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 weight: Theoretical: 990 KDa

Macromolecule #1: BcsA cellulose synthase

• Macromolecule: Name: 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 expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #2: BcsB cellulose co-polymerase

• Macromolecule: Name: 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIT DFNRVRLEFV GHYQDVCENP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPRD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SRSGWRGQNF PVLYNQLPDR NAIVFATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDAPNWVR TDRPVTFGEL KTYEEQLQSS GLEPAAINVS LNLPPDLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATNQLRFD FEYMNPMPGG SVDNCITFQP VQNHVVIGDD STIDFSKYYH FIPMPDLRAF ANAGFPFSRM ADLSQTITVM PKAPNEAQME TLLNTVGFIG AQTGFPAINL TVTDDGSTIQ GKDADIMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGAMAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLRVGDVY YVGHLPWFER LWYALANHPI LLAVLAAISV ILLAWVLWRL LRIISRRRLN PDNE

Macromolecule #3: BcsB cellulose co-polymerase

• Macromolecule: Name: BcsB cellulose co-polymerase / 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIT DFNRVRLEFV GHYQDVCENP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPRD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SRSGWRGQNF PVLYNQLPDR NAIVFATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDAPNWVR TDRPVTFGEL KTYEEQLQSS GLEPAAINVS LNLPPDLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATNQLRFD FEYMNPMPGG SVDNCITFQP VQNHVVIGDD STIDFSKYYH FIPMPDLRAF ANAGFPFSRM ADLSQTITVM PKAPNEAQME TLLNTVGFIG AQTGFPAINL TVTDDGSTIQ GKDADIMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGAMAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLRVGDVY YVGHLPWFER LWYALANHPI LLAVLAAISV ILLAWVLWRL LRIISRRRLN PDNE

Macromolecule #4: BcsB cellulose co-polymerase

• Macromolecule: Name: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 4 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIT DFNRVRLEFV GHYQDVCENP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPRD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SRSGWRGQNF PVLYNQLPDR NAIVFATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDAPNWVR TDRPVTFGEL KTYEEQLQSS GLEPAAINVS LNLPPDLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATNQLRFD FEYMNPMPGG SVDNCITFQP VQNHVVIGDD STIDFSKYYH FIPMPDLRAF ANAGFPFSRM ADLSQTITVM PKAPNEAQME TLLNTVGFIG AQTGFPAINL TVTDDGSTIQ GKDADIMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGAMAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLRVGDVY YVGHLPWFER LWYALANHPI LLAVLAAISV ILLAWVLWRL LRIISRRRLN PDNE

Macromolecule #5: BcsB cellulose co-polymerase

• Macromolecule: Name: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 5 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIT DFNRVRLEFV GHYQDVCENP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPRD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SRSGWRGQNF PVLYNQLPDR NAIVFATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDAPNWVR TDRPVTFGEL KTYEEQLQSS GLEPAAINVS LNLPPDLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATNQLRFD FEYMNPMPGG SVDNCITFQP VQNHVVIGDD STIDFSKYYH FIPMPDLRAF ANAGFPFSRM ADLSQTITVM PKAPNEAQME TLLNTVGFIG AQTGFPAINL TVTDDGSTIQ GKDADIMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGAMAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLRVGDVY YVGHLPWFER LWYALANHPI LLAVLAAISV ILLAWVLWRL LRIISRRRLN PDNE

Macromolecule #6: BcsB cellulose co-polymerase

• Macromolecule: Name: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 6 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIT DFNRVRLEFV GHYQDVCENP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPRD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SRSGWRGQNF PVLYNQLPDR NAIVFATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDAPNWVR TDRPVTFGEL KTYEEQLQSS GLEPAAINVS LNLPPDLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATNQLRFD FEYMNPMPGG SVDNCITFQP VQNHVVIGDD STIDFSKYYH FIPMPDLRAF ANAGFPFSRM ADLSQTITVM PKAPNEAQME TLLNTVGFIG AQTGFPAINL TVTDDGSTIQ GKDADIMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGAMAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLRVGDVY YVGHLPWFER LWYALANHPI LLAVLAAISV ILLAWVLWRL LRIISRRRLN PDNE

Macromolecule #7: BcsB cellulose co-polymerase

• Macromolecule: Name: BcsB cellulose co-polymerase / type: protein_or_peptide / ID: 7 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPSPSLLP VQSQLKVYLN DELMGVLPVT KEQLGKKTLA QMPINPLFIT DFNRVRLEFV GHYQDVCENP ASTTLWLDVG RSSGLDLTYQ TLNVKNDLSH FPVPFFDPRD NRTNTLPMVF AGAPDVGLQQ ASAIVASWFG SRSGWRGQNF PVLYNQLPDR NAIVFATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDAPNWVR TDRPVTFGEL KTYEEQLQSS GLEPAAINVS LNLPPDLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATNQLRFD FEYMNPMPGG SVDNCITFQP VQNHVVIGDD STIDFSKYYH FIPMPDLRAF ANAGFPFSRM ADLSQTITVM PKAPNEAQME TLLNTVGFIG AQTGFPAINL TVTDDGSTIQ GKDADIMIIG GIPDKLKDDK QIDLLVQATE SWVKTPMRQT PFPGIVPDES DRAAETRSTL TSSGAMAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLRVGDVY YVGHLPWFER LWYALANHPI LLAVLAAISV ILLAWVLWRL LRIISRRRLN PDNE

Macromolecule #8: BcsR essential cellulose secretion regulator

• Macromolecule: Name: BcsR essential cellulose secretion regulator / type: protein_or_peptide / ID: 8 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MGSSHHHHHH HHAAGSNNNE PDTLPDPAIG YIFQNDIVAL KQAFSLPDID YADISQREQL AAALKRWPLL AEFAQQK

Macromolecule #9: BcsR essential cellulose secretion regulator

• Macromolecule: Name: BcsR essential cellulose secretion regulator / type: protein_or_peptide / ID: 9 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MGSSHHHHHH HHAAGSNNNE PDTLPDPAIG YIFQNDIVAL KQAFSLPDID YADISQREQL AAALKRWPLL AEFAQQK

Macromolecule #10: BcsQ essential cellulose secretion regulator

• Macromolecule: Name: BcsQ essential cellulose secretion regulator / type: protein_or_peptide / ID: 10 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #11: BcsQ essential cellulose secretion regulator

• Macromolecule: Name: BcsQ essential cellulose secretion regulator / type: protein_or_peptide / ID: 11 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #12: BcsF cellulose secretion enhancer

• Macromolecule: Name: BcsF 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MMTISDIIEI IVVCALIFFP LGYLARHSLR RIRDTLRLFF AKPRYVKPAG TLRRTEKARA TKK

Macromolecule #13: BcsF cellulose secretion enhancer

• Macromolecule: Name: BcsF cellulose secretion enhancer / type: protein_or_peptide / ID: 13 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MMTISDIIEI IVVCALIFFP LGYLARHSLR RIRDTLRLFF AKPRYVKPAG TLRRTEKARA TKK

Macromolecule #14: BcsE cellulose secretion enhancer

• Macromolecule: Name: BcsE 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 expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #15: BcsE cellulose secretion enhancer

• Macromolecule: Name: BcsE cellulose secretion enhancer / type: protein_or_peptide / ID: 15 ; 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 expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #16: BcsG pEtN-transferase

• Macromolecule: Name: 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 expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #17: BcsG pEtN-transferase

• Macromolecule: Name: BcsG pEtN-transferase / type: protein_or_peptide / ID: 17 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #18: BcsG pEtN-transferase

• Macromolecule: Name: BcsG pEtN-transferase / type: protein_or_peptide / ID: 18 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

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

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 2 mg/mL
• Buffer: pH: 8 ; Details: 120 mM NaCL 20 mM HEPES pH8 5 mM MgCl2 10 uM ApppCp 4 uM c-di-GMP 0.01% LM-NPG
• Grid: Model: 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
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: Purification from solubilized membrane fraction using an anti-FLAG M2 affinity resin

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 20022 / Average electron dose: 49.35 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.3 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 1359795
• Startup model: Type of model: OTHER / Details: Ab Initio cryoSPARC
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: cryoSPARC / Details: Map filtered to 7A to visualize flexible regions / Number images used: 418277
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
• Final 3D classification: Number classes: 3 / Software - Name: cryoSPARC

Atomic model buiding 1

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