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- EMDB-53101: Structure of the GH13 and MucBP domains of Ruminococcus bromii Amy10 -

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Basic information

Entry
Database: EMDB / ID: EMD-53101
TitleStructure of the GH13 and MucBP domains of Ruminococcus bromii Amy10
Map data
Sample
  • Complex: Ruminococcus bromii Amy10
    • Protein or peptide: Ruminococcus bromii Amy10
KeywordsGH13 / Ruminococcus bromii / Amy10 / MucBP / HYDROLASE
Biological speciesRuminococcus bromii L2-63 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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_53101.png

Downloads & links

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Map

FileDownload / File: emd_53101.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 360 pix.
= 234.36 Å		0.65 Å/pix.
x 360 pix.
= 234.36 Å		0.65 Å/pix.
x 360 pix.
= 234.36 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: 0.026
Minimum - Maximum-0.047108088 - 0.097078435
Average (Standard dev.)0.0001881388 (±0.002889423)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 234.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53101_msk_1.map
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Additional map: Map from 3DFlex analysis in cryoSPARC

Fileemd_53101_additional_1.map
AnnotationMap from 3DFlex analysis in cryoSPARC
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Half map: #1

Fileemd_53101_half_map_1.map
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Half map: #2

Fileemd_53101_half_map_2.map
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Sample components

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Entire : Ruminococcus bromii Amy10

EntireName: Ruminococcus bromii Amy10
Components
  • Complex: Ruminococcus bromii Amy10
    • Protein or peptide: Ruminococcus bromii Amy10

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Supramolecule #1: Ruminococcus bromii Amy10

SupramoleculeName: Ruminococcus bromii Amy10 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Ruminococcus bromii L2-63 (bacteria)
Molecular weightTheoretical: 131.8 KDa

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Macromolecule #1: Ruminococcus bromii Amy10

MacromoleculeName: Ruminococcus bromii Amy10 / type: protein_or_peptide / ID: 1
Details: removed signalling peptide for secretion, added N-terminal 6x His-Tag
Number of copies: 1 / Enantiomer: LEVO / EC number: pullulanase
Source (natural)Organism: Ruminococcus bromii L2-63 (bacteria)
Molecular weightTheoretical: 131.863844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHHHHHHAV TSDESVSAGN YYNANYLESY ASKAYNESGL GSVYSKTSTT WKTWSPDASS VKLKLYTTGS DNEAGASAIG TYDMKKDSS TGVWSLNLSG DYKNKYYTYL VTVNGTTKET QDVYSQAVGV NGNRTMVVDL DSTDPSGWSD DKHVLFNSAS E AAVWEVHV ...String:
MAHHHHHHAV TSDESVSAGN YYNANYLESY ASKAYNESGL GSVYSKTSTT WKTWSPDASS VKLKLYTTGS DNEAGASAIG TYDMKKDSS TGVWSLNLSG DYKNKYYTYL VTVNGTTKET QDVYSQAVGV NGNRTMVVDL DSTDPSGWSD DKHVLFNSAS E AAVWEVHV RDFSVSKNSG VSEDNKGKYL AFAEGGTTLN SDTSSSAVST GIDYLVEQGI NCVQLMPVYD YGSVKEDVAS SS SNRNWGY DPVNYNAPEG SYSTNPYDGN TRITEFKQMI QALHDRGISV VMDVVYNHTF SNDSCFNRTV PGYYYRMHSS SAY SNGSGC GNETASDKLM YRKYMIESVK YWAEEYHIDG FRFDLMGIHD ITTMNDIRSA LDGLYSDGSG KKILMYGEPW TGGS VAISD GCSQSKAGSL NPRVGMFCDS YRDAIKGSTD GSDKGFVQGN TDKAGTVANG VTGKGFSAQA PSQTIAYADA HDNLI LWDK IVKSNGSSSW NSTSSSLRGQ VKKVMGLLLT SQGIPFMTAG SEFCRTKQGD TNSYKSSDAI NEIDWSRVKT YSDVAA YYK GLLEIRENYS PMKSSTFNTP SFQSTHGDVV AYTYSNNKSN EWGKVCVLVN ASSTNDWPIT LDGSGWTVVA DGTTAGL KS LGTVSGNTYT VPANSACVLV QSSTFNNLKV SEKTFGTVTI KHIDDSGNVL KTSTAKYADG TTYRTYPDTT ILYDYALK D TQGVTSGTVT GGKNYNVTYV YSSSGIRSGY VTVNYVDENG ESIKDTVSTK YREGDSYSVP FTSIQGYQLD TDKYPANTT GTFNGTNTTI NFVYKALDST SSVVHYYNSN NWSNVRCYAY TDGGEEPNGK WNNATVMTSE GNGWLKCTIP ASSSYVMFHT DSQQEPGAN ETGYLVSGEA WIQNKKLSFS SKVITSHIDA ATGEKIADDE ILIQSKVSSD DTYKTSPLSG RTDVIAPVNA S GNLSSGII NVVYLYTSSE RPSTAPSTVT PTTAPVTQPT EKILIGDVNL NGAIDIVDTT AVQKYIVKLI TLSDKALIAA AR CDADGEN DIVSVKDATY IQMYVAKLDG HGNVGTYYES EVTPTTAPVT EPATEEPTVA PTTVPVTTAP VTEPTTTPSS TYT VKFTDS LNWDGTLYCY SWAEDGTSTK SWPGVAMTYL NTNDYGQKVY SVEVPNTVDY IIFTNGSSQT IDIGFDGTSL NYYT ESQYD SKGHAYVGSW

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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

Concentration0.08 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: 2 / Number real images: 11102 / 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: 2138009
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: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 274000
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-9qf8:
Structure of the GH13 and MucBP domains of Ruminococcus bromii Amy10

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