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- PDB-9qf8: Structure of the GH13 and MucBP domains of Ruminococcus bromii Amy10 -

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Basic information

Entry
Database: PDB / ID: 9qf8
TitleStructure of the GH13 and MucBP domains of Ruminococcus bromii Amy10
ComponentsRuminococcus bromii Amy10
KeywordsHYDROLASE / GH13 / Ruminococcus bromii / Amy10 / MucBP
Biological speciesRuminococcus bromii L2-63 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWimmer, B.H. / Medalia, O.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation10000885 Switzerland
CitationJournal: Nat Commun / Year: 2025
Title: Spatial constraints drive amylosome-mediated resistant starch degradation by Ruminococcus bromii in the human colon.
Authors: Benedikt H Wimmer / Sarah Moraïs / Itai Amit / Omar Tovar-Herrera / Meltem Tatli / Anke Trautwein-Schult / Barbara Pfister / Ran Zalk / Paloma Tödtli / Sebastian Simoni / Matteo Lisibach / ...Authors: Benedikt H Wimmer / Sarah Moraïs / Itai Amit / Omar Tovar-Herrera / Meltem Tatli / Anke Trautwein-Schult / Barbara Pfister / Ran Zalk / Paloma Tödtli / Sebastian Simoni / Matteo Lisibach / Liron Levin / Dörte Becher / Edward A Bayer / Ohad Medalia / Itzhak Mizrahi /
Abstract: Degradation of complex dietary fiber by gut microbes is essential for colonic fermentation, short-chain fatty acid production, and microbiome function. Ruminococcus bromii is the primary resistant ...Degradation of complex dietary fiber by gut microbes is essential for colonic fermentation, short-chain fatty acid production, and microbiome function. Ruminococcus bromii is the primary resistant starch (RS) degrader in humans, which relies on the amylosome, a specialized cell-bound enzymatic complex. To unravel its architecture, function, and the interplay among its components, we applied a holistic multilayered approach: Cryo-electron tomography reveals that the amylosome comprises a constitutive extracellular layer extending toward the RS substrate. Proteomics demonstrates remodeling of its contents across different growth conditions, with Amy4 and Amy16 comprising 60% of the amylosome in response to RS. Structural and biochemical analyses reveal complementarity and synergistic RS degradation by these enzymes. We demonstrate that amylosome composition and RS degradation are regulated at two levels: structural constraints and expression-driven shifts in enzyme proportions enforce enzyme proximity, which allows R. bromii to fine-tune its adaptation to dietary fiber and shape colonic metabolism.
History
DepositionMar 11, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ruminococcus bromii Amy10


Theoretical massNumber of molelcules
Total (without water)131,8641
Polymers131,8641
Non-polymers00
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

#1: Protein Ruminococcus bromii Amy10


Mass: 131863.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: removed signalling peptide for secretion, added N-terminal 6x His-Tag
Source: (gene. exp.) Ruminococcus bromii L2-63 (bacteria) / Gene: CBL15393.1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 star / References: pullulanase
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: Ruminococcus bromii Amy10 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1318 MDa / Experimental value: NO
Source (natural)Organism: Ruminococcus bromii L2-63 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 star
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisNH2C(CH2OH)31
2137 mMsodium chlorideNaCl1
32.7 mMpotassium chlorideKCl1
410 mMcalcium chlorideCaCl21
52 mMDTTC4H10O2S21
SpecimenConc.: 0.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11102

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selectionblob picker
2EPUimage acquisition
4cryoSPARCCTF correctionblob picker
7ISOLDE1.xmodel fitting
9PHENIX1.21_5207model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2138009
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 274000 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingDetails: full-length construct / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 167.9 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00695590
ELECTRON MICROSCOPYf_angle_d0.86197593
ELECTRON MICROSCOPYf_chiral_restr0.0503836
ELECTRON MICROSCOPYf_plane_restr0.0071975
ELECTRON MICROSCOPYf_dihedral_angle_d13.87551962

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