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- PDB-9fnn: Cryo-EM structure of the c-di-GMP-saturated 'crown'less Bcs macro... -

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Basic information

Entry
Database: PDB / ID: 9fnn
TitleCryo-EM structure of the c-di-GMP-saturated 'crown'less Bcs macrocomplex for cellulose secretion in E. coli
Components
  • (Cellulose biosynthesis protein ...) x 2
  • Cell division protein
  • Cellulose synthase catalytic subunit [UDP-forming]
  • Cyclic di-GMP binding protein BcsE
  • Cyclic di-GMP-binding protein
  • Protein YhjR
KeywordsMEMBRANE PROTEIN / Bacterial cellulose secretion
Function / homology
Function and homology information


cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / cell division / plasma membrane
Similarity search - Function
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
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Chem-C2E / Cyclic di-GMP-binding protein / Cell division protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsAnso, I. / Krasteva, P.V.
Funding supportEuropean Union, 1items
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 10, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cellulose synthase catalytic subunit [UDP-forming]
B: Cyclic di-GMP-binding protein
E: Cyclic di-GMP binding protein BcsE
F: Cellulose biosynthesis protein BcsF
Q: Cell division protein
R: Protein YhjR
S: Cellulose biosynthesis protein BcsF
U: Cyclic di-GMP binding protein BcsE
V: Protein YhjR
W: Cell division protein
X: Cyclic di-GMP-binding protein
Y: Cyclic di-GMP-binding protein
Z: Cyclic di-GMP-binding protein
G: Cellulose biosynthesis protein BcsG
D: Cellulose biosynthesis protein BcsG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)783,24025
Polymers778,03515
Non-polymers5,20510
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 11 molecules ABXYZEUQWRV

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


Mass: 104015.688 Da / Num. of mol.: 1 / Mutation: HA-FLAG-tagged at C-terminue
Source method: isolated from a genetically manipulated source
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsA, yhjO, yhjP, b3533, JW5665 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: cellulose synthase (UDP-forming)
#2: Protein
Cyclic di-GMP-binding protein / Cellulose synthase regulatory subunit


Mass: 86184.383 Da / Num. of mol.: 4 / Mutation: C-terminal tail only in refined structure
Source method: isolated from a genetically manipulated source
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094
Gene: bcsB, A8C65_00280, AC789_1c39010, ACU57_05345, AM464_10380, BHS81_21120, BK292_24560, BON72_13325, BON75_10020, BON76_21165, BON94_21155, BZL31_14275, C5P01_24165, C9162_26260, CI641_010915, ...Gene: bcsB, A8C65_00280, AC789_1c39010, ACU57_05345, AM464_10380, BHS81_21120, BK292_24560, BON72_13325, BON75_10020, BON76_21165, BON94_21155, BZL31_14275, C5P01_24165, C9162_26260, CI641_010915, CWS33_18410, D3O91_11715, D9I18_15765, DQF57_09480, E0J34_09745, E0K84_22715, E2127_16410, E2128_18000, E2129_18135, E2134_17800, E5P22_13645, ELV08_08610, F1E03_18495, F1E19_10130, FRV13_18850
Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A0A061KLG7
#3: Protein Cyclic di-GMP binding protein BcsE


Mass: 60967.539 Da / Num. of mol.: 2 / Mutation: Strep-tagged at N-terminus
Source method: isolated from a genetically manipulated source
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsE, yhjS, b3536, JW3504 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)
#5: Protein Cell division protein / Cellulose biosynthesis protein BcsQ / Cellulose synthase operon protein YhjQ / Cellulose synthase / putative


Mass: 27960.832 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094
Gene: bcsQ, yhjQ, A8C65_00290, ACU57_05335, BMT91_17060, BON75_10030, BvCmsHHP019_01723, BvCmsSINP011_05061, C2U48_15650, D3O91_11725, D9H68_14440, D9I18_15755, D9I97_13990, DAH34_22885, DAH37_19450, ...Gene: bcsQ, yhjQ, A8C65_00290, ACU57_05335, BMT91_17060, BON75_10030, BvCmsHHP019_01723, BvCmsSINP011_05061, C2U48_15650, D3O91_11725, D9H68_14440, D9I18_15755, D9I97_13990, DAH34_22885, DAH37_19450, E2127_16420, E2128_18010, E2129_18145, E2134_17810, EAI42_04085, EC1094V2_71, NCTC10429_00778, NCTC11022_03734, NCTC9058_01652
Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A0A0B1KWQ0
#6: Protein Protein YhjR


Mass: 8645.541 Da / Num. of mol.: 2 / Mutation: His-tagged at N-terminus
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: yhjR, b3535, JW3503 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)

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Cellulose biosynthesis protein ... , 2 types, 4 molecules FSGD

#4: Protein Cellulose biosynthesis protein BcsF


Mass: 7378.975 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsF, Z4953, ECs4417 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)
#7: Protein Cellulose biosynthesis protein BcsG


Mass: 59687.984 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsG, yhjU, b3538, JW3506 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)

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Non-polymers , 3 types, 10 molecules

#8: Chemical
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) / c-di-GMP / Cyclic diguanosine monophosphate


Mass: 690.411 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C20H24N10O14P2 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Type: COMPLEX
Details: Locally refined c-di-GMP-bound synthase:pEtN-transferase complex from the E. coli Bcs cellulose secretion macrocomplex
Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.99 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: NiCo21(DE3)
Buffer solutionpH: 8
Details: 120 mM NaCL 20 mM HEPES pH8 5 mM MgCl2 10 uM ApppCp 4 uM c-di-GMP 0.01% LM-NPG
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purification from solubilized membrane fraction using an anti-FLAG M2 affinity resin
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm
Image recordingElectron dose: 49.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 20022
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
7Cootmodel fitting
8PHENIXmodel fitting
11cryoSPARCv4final Euler assignment
13cryoSPARCv43D reconstruction
14Cootmodel refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1359795
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 260501 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDDetailsSource nameTypeAccession code
11ColabFold model of the E. coli BcsA-BcsG2 complexOtherin silico model
21ColabFold model of the E. coli BcsA-BcsB complexOtherin silico model
31ColabFold model of the E. coli BcsE2-BcsF2 complexOtherin silico model
46yb5

6yb5

1PDBexperimental model6yb5
56ybu

6ybu

1PDBexperimental model6ybu
66ybb

6ybb

1PDBexperimental model6ybb

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