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- EMDB-50571: Cryo-EM structure of the c-di-GMP-free synthase:pEtN transferase ... -

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Basic information

Entry
Database: EMDB / ID: EMD-50571
TitleCryo-EM structure of the c-di-GMP-free synthase:pEtN transferase complex (BcsA-Bct-G3) from the E. coli cellulose secretion macrocomplex
Map dataDeep EMhancer sharpened map of the locally refined c-di-GMP-free synthase:pEtN transferase complex from the E. coli Bcs cellulose secretion system
Sample
  • Complex: Locally refined c-di-GMP-free synthase:pEtN-transferase complex from the E. coli cellulose secretion system
    • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
    • Protein or peptide: Cellulose biosynthesis protein BcsG
    • Protein or peptide: Cyclic di-GMP-binding protein
KeywordsBacterial cellulose secretion / MEMBRANE PROTEIN
Function / homologyCellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / plasma membrane / Cyclic di-GMP-binding protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.43 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_50571.png

Downloads & links

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Map

FileDownload / File: emd_50571.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeep EMhancer sharpened map of the locally refined c-di-GMP-free synthase:pEtN transferase complex from the E. coli Bcs cellulose secretion system
Projections & slices

Image control

Size
Brightness
Contrast
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

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.839 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.075
Minimum - Maximum-0.0017740906 - 2.059203
Average (Standard dev.)0.00031054346 (±0.012488798)
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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Additional map: Unsharpened map of the locally refined c-di-GMP-free synthase:pEtN...

Fileemd_50571_additional_1.map
AnnotationUnsharpened map of the locally refined c-di-GMP-free synthase:pEtN transferase complex from the E. coli Bcs cellulose secretion system
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of the locally refined c-di-GMP-free synthase:pEtN transferase...

Fileemd_50571_half_map_1.map
AnnotationHalf-map of the locally refined c-di-GMP-free synthase:pEtN transferase complex from the E. coli Bcs cellulose secretion system
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of the locally refined c-di-GMP-free synthase:pEtN transferase...

Fileemd_50571_half_map_2.map
AnnotationHalf-map of the locally refined c-di-GMP-free synthase:pEtN transferase complex from the E. coli Bcs cellulose secretion system
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Locally refined c-di-GMP-free synthase:pEtN-transferase complex f...

EntireName: Locally refined c-di-GMP-free synthase:pEtN-transferase complex from the E. coli cellulose secretion system
Components
  • Complex: Locally refined c-di-GMP-free synthase:pEtN-transferase complex from the E. coli cellulose secretion system
    • Protein or peptide: Cellulose synthase catalytic subunit [UDP-forming]
    • Protein or peptide: Cellulose biosynthesis protein BcsG
    • Protein or peptide: Cyclic di-GMP-binding protein

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Supramolecule #1: Locally refined c-di-GMP-free synthase:pEtN-transferase complex f...

SupramoleculeName: Locally refined c-di-GMP-free synthase:pEtN-transferase complex from the E. coli cellulose secretion system
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Local refinement in the context of an assembled macrocomplex
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Molecular weightTheoretical: 990 KDa

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Macromolecule #1: Cellulose synthase catalytic subunit [UDP-forming]

MacromoleculeName: Cellulose synthase catalytic subunit [UDP-forming] / type: protein_or_peptide / ID: 1
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 weightTheoretical: 104.015688 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSILTRWLLI PPVNARLIGR YRDYRRHGAS AFSATLGCFW MILAWIFIPL EHPRWQRIRA EHKNLYPHIN ASRPRPLDPV RYLIQTCWL LIGASRKETP KPRRRAFSGL QNIRGRYHQW MNELPERVSH KTQHLDEKKE LGHLSAGARR LILGIIVTFS L ILALICVT ...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

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Macromolecule #2: Cellulose biosynthesis protein BcsG

MacromoleculeName: Cellulose biosynthesis protein BcsG / type: protein_or_peptide / ID: 2
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 weightTheoretical: 59.687984 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQFTQNTAM PSSLWQYWRG LSGWNFYFLV KFGLLWAGYL NFHPLLNLVF AAFLLMPLPR YSLHRLRHWI ALPIGFALFW HDTWLPGPE SIMSQGSQVA GFSTDYLIDL VTRFINWQMI GAIFVLLVAW LFLSQWIRIT VFVVAILLWL NVLTLAGPSF S LWPAGQPT ...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

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Macromolecule #3: Cyclic di-GMP-binding protein

MacromoleculeName: Cyclic di-GMP-binding protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Molecular weightTheoretical: 83.345844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLN LEYTPSPSLL PVQSQLKVYL NDELMGVLPV TKEQLGKKTL AQMPINPLFI TDFNRVRLEF VGHYQDVCEN P ASTTLWLD ...String:
TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLN LEYTPSPSLL PVQSQLKVYL NDELMGVLPV TKEQLGKKTL AQMPINPLFI TDFNRVRLEF VGHYQDVCEN P ASTTLWLD VGRSSGLDLT YQTLNVKNDL SHFPVPFFDP RDNRTNTLPM VFAGAPDVGL QQASAIVASW FGSRSGWRGQ NF PVLYNQL PDRNAIVFAT NDKRPDFLRD HPAVKAPVIE MINHPQNPYV KLLVVFGRDD KDLLQAAKGI AQGNILFRGE SVV VNEVKP LLPRKPYDAP NWVRTDRPVT FGELKTYEEQ LQSSGLEPAA INVSLNLPPD LYLMRSTGID MDINYRYTMP PVKD SSRMD ISLNNQFLQS FNLSSKQEAN RLLLRIPVLQ GLLDGKTDVS IPALKLGATN QLRFDFEYMN PMPGGSVDNC ITFQP VQNH VVIGDDSTID FSKYYHFIPM PDLRAFANAG FPFSRMADLS QTITVMPKAP NEAQMETLLN TVGFIGAQTG FPAINL TVT DDGSTIQGKD ADIMIIGGIP DKLKDDKQID LLVQATESWV KTPMRQTPFP GIVPDESDRA AETRSTLTSS GAMAAVI GF QSPYNDQRSV IALLADSPRG YEMLNDAVND SGKRATMFGS VAVIRESGIN SLRVGDVYYV GHLPWFERLW YALANHPI L LAVLAAISVI LLAWVLWRLL RIISRRRLNP DNE

UniProtKB: Cyclic di-GMP-binding protein

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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
DetailsPurified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3

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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

DetailsGIF Quantum LS
Particle selectionNumber selected: 1359795
Startup modelType of model: INSILICO MODEL
In silico model: ColabFold model for E. coli BcsA-BcsG3 complex
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4) / Number images used: 314979
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 (ver. v4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

fold


Chain - Source name: Other / Chain - Initial model type: in silico model / Details: ColabFold model for full-length BcsA-BcsG3 complex
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9fmv:
Cryo-EM structure of the c-di-GMP-free synthase:pEtN transferase complex (BcsA-Bct-G3) from the E. coli cellulose secretion macrocomplex

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