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- EMDB-50619: Cryo-EM structure of the BcsE2F2 regulatory subcomplex from the E... -

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Basic information

Entry
Database: EMDB / ID: EMD-50619
TitleCryo-EM structure of the BcsE2F2 regulatory subcomplex from the E. coli Bcs macrocomplex for cellulose secretion (local refinement)
Map dataDeep EMhancer sharpened map from local refinement with mask around the BcsE2F2 complex from the c-di-GMP-saturated Bcs macrocomplex for E. coli cellulose secretion
Sample
  • Complex: Locally refined BcsE2F2 regulatory subcomplex in saturating c-di-GMP
    • Protein or peptide: Cyclic di-GMP binding protein BcsE
    • 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)
KeywordsBacterial cellulose secretion / MEMBRANE PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 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 11, 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_50619.png

Downloads & links

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Map

FileDownload / File: emd_50619.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeep EMhancer sharpened map from local refinement with mask around the BcsE2F2 complex from the c-di-GMP-saturated Bcs macrocomplex for E. coli cellulose secretion
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-0.0017975564 - 2.0163853
Average (Standard dev.)0.00042887303 (±0.014194279)
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 from local refinement with mask around...

Fileemd_50619_additional_1.map
AnnotationUnsharpened map from local refinement with mask around the BcsE2F2 complex from the c-di-GMP-saturated Bcs macrocomplex for E. coli cellulose secretion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map from local refinement with mask around the...

Fileemd_50619_half_map_1.map
AnnotationHalf-map from local refinement with mask around the BcsE2F2 complex from the c-di-GMP-saturated Bcs macrocomplex for E. coli cellulose secretion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map from local refinement with mask around the...

Fileemd_50619_half_map_2.map
AnnotationHalf-map from local refinement with mask around the BcsE2F2 complex from the c-di-GMP-saturated Bcs macrocomplex for E. coli cellulose secretion.
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 BcsE2F2 regulatory subcomplex in saturating c-di-GMP

EntireName: Locally refined BcsE2F2 regulatory subcomplex in saturating c-di-GMP
Components
  • Complex: Locally refined BcsE2F2 regulatory subcomplex in saturating c-di-GMP
    • Protein or peptide: Cyclic di-GMP binding protein BcsE
    • 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)

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Supramolecule #1: Locally refined BcsE2F2 regulatory subcomplex in saturating c-di-GMP

SupramoleculeName: Locally refined BcsE2F2 regulatory subcomplex in saturating c-di-GMP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Locally refined BcsE2F2 subcomplex in the context of the c-di-GMP-saturated Bcs macrocomplex for cellulose secretion in E. coli
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Molecular weightTheoretical: 990 KDa

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Macromolecule #1: Cyclic di-GMP binding protein BcsE

MacromoleculeName: 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 weightTheoretical: 60.967539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASWSHPQFE KGSMRDIVDP VFSIGISSLW DELRHMPAGG VWWFNVDRHE DAISLANQTI ASQAETAHVA VISMDSDPAK IFQLDDSQG PEKIKLFSML NHEKGLYYLT RDLQCSIDPH NYLFILVCAN NAWQNIPAER LRSWLDKMNK WSRLNHCSLL V INPGNNND ...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

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Macromolecule #2: Cellulose biosynthesis protein BcsF

MacromoleculeName: Cellulose biosynthesis protein BcsF / 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 weightTheoretical: 7.378975 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MMTISDIIEI IVVCALIFFP LGYLARHSLR RIRDTLRLFF AKPRYVKPAG TLRRTEKARA TKK

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

MacromoleculeName: 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: 3 / Number of copies: 4 / Formula: C2E
Molecular weightTheoretical: 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)

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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
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: EMDB MAP
EMDB ID:

50599


Details: The Bcs macrocomplex map in saturating c-di-GMP
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4) / Number images used: 260501
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4)
Final 3D classificationNumber classes: 3
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainDetailsPDB ID
source_name: Other, initial_model_type: in silico modelColabFold model of the E. coli BcsE2-BcsF2 complex
source_name: PDB, initial_model_type: experimental model

6ybu

source_name: PDB, initial_model_type: experimental model

6ybb

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9fo7:
Cryo-EM structure of the BcsE2F2 regulatory subcomplex from the E. coli Bcs macrocomplex for cellulose secretion (local refinement)

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