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- EMDB-53097: Structure of the GH13 domain of Ruminococcus bromii Amy4 -

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Basic information

Entry
Database: EMDB / ID: EMD-53097
TitleStructure of the GH13 domain of Ruminococcus bromii Amy4
Map data
Sample
  • Complex: Ruminococcus bromii Amy4
    • Protein or peptide: Ruminococcus bromii Amy4
KeywordsGH13 / Ruminococcus bromii / Amy4 / HYDROLASE
Biological speciesRuminococcus bromii L2-63 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsWimmer BH / Medalia O
Funding support Switzerland, 1 items
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, 2025-
Header (metadata) releaseOct 22, 2025-
Map releaseOct 22, 2025-
UpdateDec 10, 2025-
Current statusDec 10, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53097.png

Downloads & links

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Map

FileDownload / File: emd_53097.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 384 pix.
= 249.984 Å		0.65 Å/pix.
x 384 pix.
= 249.984 Å		0.65 Å/pix.
x 384 pix.
= 249.984 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.651 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.060244016 - 0.14091127
Average (Standard dev.)-0.00013755383 (±0.0027198505)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 249.98401 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53097_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_53097_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_53097_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Ruminococcus bromii Amy4

EntireName: Ruminococcus bromii Amy4
Components
  • Complex: Ruminococcus bromii Amy4
    • Protein or peptide: Ruminococcus bromii Amy4

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Supramolecule #1: Ruminococcus bromii Amy4

SupramoleculeName: Ruminococcus bromii Amy4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Ruminococcus bromii L2-63 (bacteria)
Molecular weightTheoretical: 135.6 KDa

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Macromolecule #1: Ruminococcus bromii Amy4

MacromoleculeName: Ruminococcus bromii Amy4 / type: protein_or_peptide / ID: 1
Details: removal of secretion peptide and dockerin domain, addition of a C-terminal 6x-His Tag
Number of copies: 1 / Enantiomer: LEVO / EC number: alpha-amylase
Source (natural)Organism: Ruminococcus bromii L2-63 (bacteria) / Strain: L2-63
Molecular weightTheoretical: 135.745984 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGATVSDDSS TSASSTAAGS LVEDDGFTWD NANVYFLLTD RFKNGNTAND HSYGRTLDKD GKPLEGWDTN PGTFHGGDFA GVTQSIEEG YFDNLGVNAI WISAPYEQTH GYCDSGKGFA HYSYHGYYVL DYTETDANFG TKEEFKTLVD TAHEHGIRVI M DIVMNHTG ...String:
MGATVSDDSS TSASSTAAGS LVEDDGFTWD NANVYFLLTD RFKNGNTAND HSYGRTLDKD GKPLEGWDTN PGTFHGGDFA GVTQSIEEG YFDNLGVNAI WISAPYEQTH GYCDSGKGFA HYSYHGYYVL DYTETDANFG TKEEFKTLVD TAHEHGIRVI M DIVMNHTG YNTVADMEQF GFGTLLDGAL DFKYKLTDVG EVNDHIDYKT SEEDWGKWWS NDWIRSGLPG YTEGAGGDLT MS LSGLPDF RTEQTKDVTI PPILKTKWTQ EGTYAQKLAK YGDKNTVTGY LSTWLSEWVR EYGVDGFRCD TAKHVDKASW NQL KQACVS ALREWRSNNK GKAGADWKED FWMTGEHWDH GVGYDTYYSE GGFDSMINFA VTGGGALASG SVANTYQGYA DKIN TKEGF NVLSFMSSHD ETLTRGDENT MLYNAAAFLL LPGGVQIFYG DETNRPLYPG VAFDGYGGSG HALRSDMNWN DMDKT LLAQ WQKVGQFRNK HVSIGAGANV KLSATSGVAF GRTYDKNGVT DQAAAVIGCN KNSSVTVDVS TLWEDGSYVI NTYDNS SSV VTDGKVTFNS GVNGTILMEN ADGQPLVSIT GEPKFYGTEQ VTVSLKECDS AKVSVDGGNK FVVKDGDTFT IGQTAYK ND TIEVTVEATN DKASSEAKFT FLKLGENDEK PTTSPTQSTT VPGSTASQTS KLTIKSTAGA PNVYAWTGAS TALNGAWP G QKAAAVSGQA NTYELTLPVS ADKSFSVVLN NGSGSQSGDI TGLYDGAVLE IQNGNYASVK TVTNGNQQGG GGEPEPAEG SVSVTIKPYS PSASYKIYAW NDTQKLTGAW PGVALTEKDS DGNYVVKFDN VDEVSIILSS GSGQTADITG VRDGATIEIT NEGCTTYKL TSKPIVVSPY ESLKKEARKI LAMTASDYTA ESWANAQKVL KSAEAMIKAG EDATTAEDMN AMIADLKSAQ K ALVLAPAT LTYAVVGKSV VSGVTASAAK VTVTVDGKTY TATADDVTGA FTVATSALKG TSTIKVDATR NGVNGTYSYA MK NGNIENG FVPTSPTLPT QPTTSTQPTT ATQPTTATQP TAPTQPTTAT EPTTAPAQKL TVNATSNYFG SASKTVSTDG KKV TVLYKL NSNMGVVDGQ WAVKYDSSKL KFNMADNKAD GKQTIMPSQS NLIHNAYSNV IKGVFTNPQV PTQYNGDTFI SLTF EVIGS GTASVYLDTQ YLTLGYVKGN KLNQASVVNN SKVCDVTGIA GFEKVSVNAS TAFVGDVTHH HHHH

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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

Concentration0.3 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMNH2C(CH2OH)3Tris
137.0 mMNaClsodium chloride
2.7 mMKClpotassium chloride
10.0 mMCaCl2calcium chloride
2.0 mMC4H10O2S2DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 25000 / Average electron dose: 65.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 16700000
CTF correctionSoftware - Name: cryoSPARC / Software - details: blob picker / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: cryoSPARC ab initio
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 905000
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: Non-uniform refinement
Final angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: Non-uniform refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: full-length construct
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9qf3:
Structure of the GH13 domain of Ruminococcus bromii Amy4

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