EMDB-50632: Cryo-EM structure of the 'crown'less Bcs macrocomplex for E. coli...

Basic information

Entry

Database: EMDB / ID: EMD-50632

• Title: Cryo-EM structure of the 'crown'less Bcs macrocomplex for E. coli cellulose secretion in non-saturating c-di-GMP (local)
• Map data: Deep EMhancer-sharpened cryo-EM map of the 'crown'less Bcs macrocomplex for cellulose secretion in E. coli in non-saturating c-di-GMP (local)

Sample

• Complex: Locally refined 'crown'less Bcs macrocomplex in non-saturating c-di-GMP from the E. coli cellulose secretion system
  • Protein or peptide: Cyclic di-GMP binding protein BcsE
  • Protein or peptide: Cell division protein
  • Protein or peptide: Protein YhjR
  • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
  • Protein or peptide: Cellulose biosynthesis protein BcsG
  • Protein or peptide: Cyclic di-GMP-binding protein
  • Protein or peptide: Cellulose biosynthesis protein BcsF
• Ligand: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
• Ligand: ADENOSINE-5'-TRIPHOSPHATE
• Ligand: MAGNESIUM ION

• Keywords: Bacterial cellulose secretion / MEMBRANE PROTEIN

Function / homology

Function:

cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / negative regulation of cell division / cytoplasmic side of plasma membrane / cell division / ATP hydrolysis activity / ATP binding / plasma membrane / cytosol

Domain/homology:

Cellulose synthase operon protein BcsQ / Cellulose biosynthesis protein BcsQ / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / : / P-loop containing nucleoside triphosphate hydrolase

Component:

Cyclic di-GMP-binding protein / Cell division protein

• Biological species: Escherichia coli (E. coli)
• Method: single particle reconstruction / cryo EM / Resolution: 3.37 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 12, 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_50632.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Deep EMhancer-sharpened cryo-EM map of the 'crown'less Bcs macrocomplex for cellulose secretion in E. coli in non-saturating c-di-GMP (local)
• Voxel size: X=Y=Z: 0.839 Å

Density

Contour Level	By AUTHOR: 0.025
Minimum - Maximum	-0.0017767221 - 1.7456958
Average (Standard dev.)	0.00058847404 (±0.014503919)

• 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

Mask #1

• File: emd_50632_msk_1.map

Additional map: Unsharpened cryo-EM map of the 'crown'less Bcs macrocomplex...

• File: emd_50632_additional_1.map
• Annotation: Unsharpened cryo-EM map of the 'crown'less Bcs macrocomplex for cellulose secretion in E. coli in non-saturating c-di-GMP (local)

Half map: Half-map of the 'crown'less Bcs macrocomplex for cellulose...

• File: emd_50632_half_map_1.map
• Annotation: Half-map of the 'crown'less Bcs macrocomplex for cellulose secretion in E. coli in non-saturating c-di-GMP (local)

Half map: Half-map of the 'crown'less Bcs macrocomplex for cellulose...

• File: emd_50632_half_map_2.map
• Annotation: Half-map of the 'crown'less Bcs macrocomplex for cellulose secretion in E. coli in non-saturating c-di-GMP (local)

Sample components

Entire : Locally refined 'crown'less Bcs macrocomplex in non-saturating c-...

• Entire: Name: Locally refined 'crown'less Bcs macrocomplex in non-saturating c-di-GMP from the E. coli cellulose secretion system

Components

• Complex: Locally refined 'crown'less Bcs macrocomplex in non-saturating c-di-GMP from the E. coli cellulose secretion system
  • Protein or peptide: Cyclic di-GMP binding protein BcsE
  • Protein or peptide: Cell division protein
  • Protein or peptide: Protein YhjR
  • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
  • Protein or peptide: Cellulose biosynthesis protein BcsG
  • Protein or peptide: Cyclic di-GMP-binding protein
  • Protein or peptide: Cellulose biosynthesis protein BcsF
• Ligand: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
• Ligand: ADENOSINE-5'-TRIPHOSPHATE
• Ligand: MAGNESIUM ION

Supramolecule #1: Locally refined 'crown'less Bcs macrocomplex in non-saturating c-...

• Supramolecule: Name: Locally refined 'crown'less Bcs macrocomplex in non-saturating c-di-GMP from the E. coli cellulose secretion system; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7; Details: Local refinement in the context of an assembled macrocomplex
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 990 KDa

Macromolecule #1: Cyclic di-GMP binding protein BcsE

• Macromolecule: Name: Cyclic di-GMP binding protein BcsE / type: protein_or_peptide / ID: 1 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 60.967539 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MASWSHPQFE KGSMRDIVDP VFSIGISSLW DELRHMPAGG VWWFNVDRHE DAISLANQTI ASQAETAHVA VISMDSDPAK IFQLDDSQG PEKIKLFSML NHEKGLYYLT RDLQCSIDPH NYLFILVCAN NAWQNIPAER LRSWLDKMNK WSRLNHCSLL V INPGNNND KQFSLLLEEY RSLFGLASLR FQGDQHLLDI AFWCNEKGVS ARQQLSVQQQ NGIWTLVQSE EAEIQPRSDE KR ILSNVAV LEGAPPLSEH WQLFNNNEVL FNEARTAQAA TVVFSLQQNA QIEPLARSIH TLRRQRGSAM KILVRENTAS LRA TDERLL LACGANMVIP WNAPLSRCLT MIESVQGQKF SRYVPEDITT LLSMTQPLKL RGFQKWDVFC NAVNNMMNNP LLPA HGKGV LVALRPVPGI RVEQALTLCR PNRTGDIMTI GGNRLVLFLS FCRINDLDTA LNHIFPLPTG DIFSNRMVWF EDDQI SAEL VQMRLLAPEQ WGMPLPLTQS SKPVINAEHD GRHWRRIPEP MRLLDDAVER SS

Macromolecule #2: Cell division protein

• Macromolecule: Name: Cell division protein / type: protein_or_peptide / ID: 2 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 27.960832 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MAVLGLQGVR GGVGTTTITA ALAWSLQMLG ENVLVVDACP DNLLRLSFNV DFTHRQGWAR AMLDGQDWRD AGLRYTSQLD LLPFGQLSI EEQENPQHWQ TRLSDICSGL QQLKASGRYQ WILIDLPRDA SQITHQLLSL CDHSLAIVNV DANCHIRLHQ Q ALPDGAHI LINNFRIGSQ VQDDIYQLWL QSQRRLLPML IHRDEAMAEC LAAKQPVGEY RSDALAAEEI LTLANWCLLN YS GLKTPVG SKS

UniProtKB: Cell division protein

Macromolecule #3: Protein YhjR

• Macromolecule: Name: Protein YhjR / type: protein_or_peptide / ID: 3 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 8.645541 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MGSSHHHHHH HHAAGSNNNE PDTLPDPAIG YIFQNDIVAL KQAFSLPDID YADISQREQL AAALKRWPLL AEFAQQK

Macromolecule #4: Cellulose synthase catalytic subunit [UDP-forming]

• Macromolecule: Name: Cellulose synthase catalytic subunit [UDP-forming] / type: protein_or_peptide / ID: 4 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 1 / Enantiomer: LEVO / EC number: cellulose synthase (UDP-forming)
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 104.015688 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWL LIGASRKETP KPRRRAFSGL QNIRGRYHQW MNELPERVSH KTQHLDEKKE LGHLSAGARR LILGIIVTFS L ILALICVT QPFNPLAQFI FLMLLWGVAL IVRRMPGRFS ALMLIVLSLT VSCRYIWWRY TSTLNWDDPV SLVCGLILLF AE TYAWIVL VLGYFQVVWP LNRQPVPLPK DMSLWPSVDI FVPTYNEDLN VVKNTIYASL GIDWPKDKLN IWILDDGGRE EFR QFAQNV GVKYIARTTH EHAKAGNINN ALKYAKGEFV SIFDCDHVPT RSFLQMTMGW FLKEKQLAMM QTPHHFFSPD PFER NLGRF RKTPNEGTLF YGLVQDGNDM WDATFFCGSC AVIRRKPLDE IGGIAVETVT EDAHTSLRLH RRGYTSAYMR IPQAA GLAT ESLSAHIGQR IRWARGMVQI FRLDNPLTGK GLKFAQRLCY VNAMFHFLSG IPRLIFLTAP LAFLLLHAYI IYAPAL MIA LFVLPHMIHA SLTNSKIQGK YRHSFWSEIY ETVLAWYIAP PTLVALINPH KGKFNVTAKG GLVEEEYVDW VISRPYI FL VLLNLVGVAV GIWRYFYGPP TEMLTVVVSM VWVFYNLIVL GGAVAVSVES KQVRRSHRVE MTMPAAIARE DGHLFSCT V QDFSDGGLGI KINGQAQILE GQKVNLLLKR GQQEYVFPTQ VARVMGNEVG LKLMPLTTQQ HIDFVQCTFA RADTWALWQ DSYPEDKPLE SLLDILKLGF RGYRHLAEFA PSSVKGIFRV LTSLVSWVVS FIPRRPERSE TAQPSDQALA QQGSARSSGR TGLEFEEFY PYDVPDYAAD YKDDDDKRS

Macromolecule #5: Cellulose biosynthesis protein BcsG

• Macromolecule: Name: Cellulose biosynthesis protein BcsG / type: protein_or_peptide / ID: 5 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 59.687984 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPE SIMSQGSQVA GFSTDYLIDL VTRFINWQMI GAIFVLLVAW LFLSQWIRIT VFVVAILLWL NVLTLAGPSF S LWPAGQPT TTVTTTGGNA AATVAATGGA PVVGDMPAQT APPTTANLNA WLNNFYNAEA KRKSTFPSSL PADAQPFELL VI NICSLSW SDIEAAGLMS HPLWSHFDIE FKNFNSATSY SGPAAIRLLR ASCGQTSHTN LYQPANNDCY LFDNLSKLGF TQH LMMGHN GQFGGFLKEV RENGGMQSEL MDQTNLPVIL LGFDGSPVYD DTAVLNRWLD VTEKDKNSRS ATFYNTLPLH DGNH YPGVS KTADYKARAQ KFFDELDAFF TELEKSGRKV MVVVVPEHGG ALKGDRMQVS GLRDIPSPSI TDVPVGVKFF GMKAP HQGA PIVIEQPSSF LAISDLVVRV LDGKIFTEDN VDWKKLTSGL HKQHRSPRTQ MQ

Macromolecule #6: Cyclic di-GMP-binding protein

• Macromolecule: Name: Cyclic di-GMP-binding protein / type: protein_or_peptide / ID: 6 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 86.184383 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVL RGINPNGSIE FGMRSDEVVT KAMLNLEYTP SPSLLPVQSQ LKVYLNDELM GVLPVTKEQL GKKTLAQMPI N PLFITDFN RVRLEFVGHY QDVCENPAST TLWLDVGRSS GLDLTYQTLN VKNDLSHFPV PFFDPRDNRT NTLPMVFAGA PD VGLQQAS AIVASWFGSR SGWRGQNFPV LYNQLPDRNA IVFATNDKRP DFLRDHPAVK APVIEMINHP QNPYVKLLVV FGR DDKDLL QAAKGIAQGN ILFRGESVVV NEVKPLLPRK PYDAPNWVRT DRPVTFGELK TYEEQLQSSG LEPAAINVSL NLPP DLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATN QLRF DFEYMNPMPG GSVDNCITFQ PVQNHVVIGD DSTIDFSKYY HFIPMPDLRA FANAGFPFSR MADLSQTITV MPKAPN EAQ METLLNTVGF IGAQTGFPAI NLTVTDDGST IQGKDADIMI IGGIPDKLKD DKQIDLLVQA TESWVKTPMR QTPFPGI VP DESDRAAETR STLTSSGAMA AVIGFQSPYN DQRSVIALLA DSPRGYEMLN DAVNDSGKRA TMFGSVAVIR ESGINSLR V GDVYYVGHLP WFERLWYALA NHPILLAVLA AISVILLAWV LWRLLRIISR RRLNPDNE

UniProtKB: Cyclic di-GMP-binding protein

Macromolecule #7: Cellulose biosynthesis protein BcsF

• Macromolecule: Name: Cellulose biosynthesis protein BcsF / type: protein_or_peptide / ID: 7 ; Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: 1094
• Molecular weight: Theoretical: 7.378975 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MMTISDIIEI IVVCALIFFP LGYLARHSLR RIRDTLRLFF AKPRYVKPAG TLRRTEKARA TKK

Macromolecule #8: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydr...

• Macromolecule: Name: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one); type: ligand / ID: 8 / Number of copies: 4 / Formula: C2E
• Molecular weight: Theoretical: 690.411 Da

Chemical component information

ChemComp-C2E:
9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)

Macromolecule #9: ADENOSINE-5'-TRIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 9 / Number of copies: 2 / Formula: ATP
• Molecular weight: Theoretical: 507.181 Da

Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

Macromolecule #10: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

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
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K
• Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3

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
• Sample stage: Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: GIF Quantum LS
• Particle selection: Number selected: 1359795
• Startup model: Type of model: INSILICO MODEL; In silico model: Ab-initio and 3D classification (cryoSPARC)
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4) / Number images used: 314979
• 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 (ver. v4)

Atomic model buiding 1

Initial model

PDB ID	Chain	Details
fold	source_name: Other, initial_model_type: in silico model	ColabFold model for full-length BcsA-BcsG3 complex
6ybb	source_name: PDB, initial_model_type: experimental model	crystal structure of the BcsERQ complex
fold	source_name: Other, initial_model_type: in silico model	ColabFold model for full-length BcsE2-BcsF2 complex

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Output model

PDB-9fp0:
Cryo-EM structure of the 'crown'less Bcs macrocomplex for E. coli cellulose secretion in non-saturating c-di-GMP (local)