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- EMDB-77065: CryoEM structure of AdhE spirosome from Clostridium thermocellum ... -

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Basic information

Entry
Database: EMDB / ID: EMD-77065
TitleCryoEM structure of AdhE spirosome from Clostridium thermocellum uncovered by visual proteomics.
Map dataSharpened cryoEM map of Ct spirosome obtained by helical refinement in cryoSPARC
Sample
  • Complex: AdhE spirosome
    • Protein or peptide: Aldehyde-alcohol dehydrogenase
  • Ligand: FE (III) ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
KeywordsDehydrogenase / Spirosome / Filament / OXIDOREDUCTASE
Function / homology
Function and homology information


butanol dehydrogenase (NAD+) activity / acetaldehyde dehydrogenase (acetylating) activity / methylglyoxal reductase (NADPH) (acetol producing) activity / alcohol metabolic process / carbon utilization / alcohol dehydrogenase (NADP+) activity / aldehyde dehydrogenase (NAD+) activity / metal ion binding / cytosol
Similarity search - Function
Butanol dehydrogenase-like / Bifunctional aldehyde-alcohol dehydrogenase / Bifunctional aldehyde-alcohol dehydrogenase, C-terminal domain / Iron-containing alcohol dehydrogenases signature 2. / Iron-containing alcohol dehydrogenases signature 1. / : / Fe-containing alcohol dehydrogenase family, C-terminal / Alcohol dehydrogenase, iron-type, conserved site / Alcohol dehydrogenase, iron-type/glycerol dehydrogenase GldA / Iron-containing alcohol dehydrogenase ...Butanol dehydrogenase-like / Bifunctional aldehyde-alcohol dehydrogenase / Bifunctional aldehyde-alcohol dehydrogenase, C-terminal domain / Iron-containing alcohol dehydrogenases signature 2. / Iron-containing alcohol dehydrogenases signature 1. / : / Fe-containing alcohol dehydrogenase family, C-terminal / Alcohol dehydrogenase, iron-type, conserved site / Alcohol dehydrogenase, iron-type/glycerol dehydrogenase GldA / Iron-containing alcohol dehydrogenase / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, C-terminal / Aldehyde dehydrogenase, N-terminal / Aldehyde/histidinol dehydrogenase
Similarity search - Domain/homology
Aldehyde-alcohol dehydrogenase
Similarity search - Component
Biological speciesAcetivibrio thermocellus DSM 1313 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 4.07 Å
AuthorsAgdanowski MP / Rodriguez JA / Moser T / Evans JE
Funding support United States, 2 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: bioRxiv / Year: 2026
Title: Visual exoproteomics of during anaerobic biomass-degradation identifies functional spirosomes.
Authors: Matthew P Agdanowski / Matthew J Kensil / Trevor H Moser / Ethan Humm / Yaneli I Guandique / Kayleigh Mason-Chalmers / Tracy Al-Set / Rachel R Ogorzalek Loo / James E Evans / Robert P ...Authors: Matthew P Agdanowski / Matthew J Kensil / Trevor H Moser / Ethan Humm / Yaneli I Guandique / Kayleigh Mason-Chalmers / Tracy Al-Set / Rachel R Ogorzalek Loo / James E Evans / Robert P Gunsalus / Joseph A Loo / Jose A Rodriguez
Abstract: Visual proteomics enables the study of low-abundance proteins and identification of unknown complexes from heterogeneous samples by complementing high-resolution cryogenic electron microscopy (cryoEM) ...Visual proteomics enables the study of low-abundance proteins and identification of unknown complexes from heterogeneous samples by complementing high-resolution cryogenic electron microscopy (cryoEM) with external inputs on protein identity such as mass spectrometry. Using this approach, we interrogated the exoproteome of the anaerobic cellulose-degrading bacterium as it carried out biomass degradation. Mass spectrometry indicated a broad exoproteome composition, including cellulose degrading machinery CelA and CipA. A focus on large exoproteome assemblies revealed abundant protein filaments and pleomorphic vesicular structures. Analysis of the most abundant protein filaments yielded an ∼4 □ resolution native structure that, aided by mass spectrometry, modeling, and structural searching, was found to be the aldehyde-alcohol dehydrogenase (AdhE) spirosome. AdhE contained both NAD and Fe in their expected binding sites and biochemical and structural analyses of enriched spirosome preparations indicated they were functional. Altered NADH solution concentrations triggered conformational changes in the exoproteomic spirosomes, and the constituent AdhE remained capable of ethanol production. Although the basis for functional extracellular spirosome accumulation in live anaerobic cultures remains unclear, their abundance in crude exoproteomes suggests their presence could influence biomass fueled growth.
History
DepositionMay 6, 2026-
Header (metadata) releaseJun 24, 2026-
Map releaseJun 24, 2026-
UpdateJun 24, 2026-
Current statusJun 24, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_77065.png

Downloads & links

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Map

FileDownload / File: emd_77065.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryoEM map of Ct spirosome obtained by helical refinement in cryoSPARC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 512 pix.
= 348.16 Å		0.68 Å/pix.
x 512 pix.
= 348.16 Å		0.68 Å/pix.
x 512 pix.
= 348.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.081321776 - 0.18744588
Average (Standard dev.)0.00064235827 (±0.008742937)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 348.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened cryoEM map of Ct spirosome obtained by...

Fileemd_77065_additional_1.map
AnnotationUnsharpened cryoEM map of Ct spirosome obtained by helical refinement in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A of helical Ct Spirosome

Fileemd_77065_half_map_1.map
AnnotationHalf Map A of helical Ct Spirosome
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B of helical Ct Spirosome

Fileemd_77065_half_map_2.map
AnnotationHalf Map B of helical Ct Spirosome
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AdhE spirosome

EntireName: AdhE spirosome
Components
  • Complex: AdhE spirosome
    • Protein or peptide: Aldehyde-alcohol dehydrogenase
  • Ligand: FE (III) ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: AdhE spirosome

SupramoleculeName: AdhE spirosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Acetivibrio thermocellus DSM 1313 (bacteria)

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Macromolecule #1: Aldehyde-alcohol dehydrogenase

MacromoleculeName: Aldehyde-alcohol dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Acetivibrio thermocellus DSM 1313 (bacteria)
Molecular weightTheoretical: 94.788023 KDa
SequenceString: EVIDNVEKLE KALKRLREAQ SVYATYTQEQ VDKIFFEAAM AANKMRIPLA KMAVEETGMG VVEDKVIKNH YASEYIYNAY KNTKTCGVI EEDPAFGIKK IAEPLGVIAA VIPTTNPTST AIFKTLIALK TRNAIIISPH PRAKNSTIEA AKIVLEAAVK A GAPEGIIG ...String:
EVIDNVEKLE KALKRLREAQ SVYATYTQEQ VDKIFFEAAM AANKMRIPLA KMAVEETGMG VVEDKVIKNH YASEYIYNAY KNTKTCGVI EEDPAFGIKK IAEPLGVIAA VIPTTNPTST AIFKTLIALK TRNAIIISPH PRAKNSTIEA AKIVLEAAVK A GAPEGIIG WIDVPSLELT NLVMREADVI LATGGPGLVK AAYSSGKPAI GVGAGNTPAI IDDSADIVLA VNSIIHSKTF DN GMICASE QSVIVLDGVY KEVKKEFEKR GCYFLNEDET EKVRKTIIIN GALNAKIVGQ KAHTIANLAG FEVPETTKIL IGE VTSVDI SEEFAHEKLC PVLAMYRAKD FDDALDKAER LVADGGFGHT SSLYIDTVTQ KEKLQKFSER MKTCRILVNT PSSQ GGIGD LYNFKLAPSL TLGCGSWGGN SVSDNVGVKH LLNIKTVAER RENMLWFRTP EKIYIKRGCL PVALDELKNV MGKKK AFIV TDNFLYNNGY TKPITDKLDE MGIVHKTFFD VSPDPSLASA KAGAAEMLAF QPDTIIAVGG GSAMDAAKIM WVMYEH PEV DFMDMAMRFM DIRKRVYTFP KMGQKAYFIA IPTSAGTGSE VTPFAVITDE KTGIKYPLAD YELLPDMAIV DADMMMN AP KGLTAASGID ALTHALEAYV SMLATDYTDS LALRAIKMIF EYLPRAYENG ASDPVAREKM ANAATIAGMA FANAFLGV C HSMAHKLGAF YHLPHGVANA LMINEVIRFN SSEAPTKMGT FPQYDHPRTL ERYAEIADYI GLKGKNNEEK VENLIKAID ELKEKVGIRK TIKDYDIDEK EFLDRLDEMV EQAFDDQCTG TNPRYPLMNE IRQMYLNAYY G

UniProtKB: Aldehyde-alcohol dehydrogenase

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Macromolecule #2: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #3: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 6 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
150.0 mMNaClsodium chloride
2.0 mMCaCl2calcium chloride

Details: 20mM Tris pH 8.0, 150mM NaCl, 2mM CaCl2
GridModel: EMS Formvar Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
Details: CFlat 1.2/1.3 300 mesh holey carbon Cu support grids were negatively glow discharged for 20 seconds on the sample side.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 285.15 K / Instrument: FEI VITROBOT MARK IV
Details: Vitification was performed on CFlat 1.2/1.3 300 mesh holey carbon grids with copper support..
DetailsSample was obtained from partially-purified extracellular media and contained an array of unidentified species.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 88184 pixel / Number grids imaged: 1 / Number real images: 4863 / Average exposure time: 1.03 sec. / Average electron dose: 48.0 e/Å2
Details: Images were recorded as movies consisting of 50 frames over an exposure time of 1.03 seconds, with a total accumulated dose of 48 electrons per Angstrom
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 65
Applied symmetry - Helical parameters - Δz: 60.06 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.03 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.07 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1)
Software - details: Helical Refinement, Local Refinement with mask
Details: Overall resolution 4.07 angstrom as estimated by the masked gold-standard FSC = 0.143 criterion in cryoSPARC. Unmasked FSC values are lower, consistent with expected effects of masking." in the details field.
Number images used: 95407
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Software - details: Patch CTF Estimation / Type: NONE
Startup modelType of model: NONE
Details: Initial model was generated by running an Ab Initio model job on particles from the selected 2D classes
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 4.4.1)
Software - details: Helical Refinement with optimized helical rise and twist
Details: Final particle orientations were refined during cryoSPARC helical refinement. Initial helical refinement was performed without imposed helical parameters. Helical symmetry was subsequently ...Details: Final particle orientations were refined during cryoSPARC helical refinement. Initial helical refinement was performed without imposed helical parameters. Helical symmetry was subsequently estimated using cryoSPARC Helical Symmetry Search and refined in later helical refinement jobs with rise and twist allowed to optimize.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8uhw


Chain - Chain ID: A / Chain - Residue range: 11-869 / Chain - Source name: PDB / Chain - Initial model type: experimental model
Details: published structure was fast relaxed and then used as an initial model for model building and refinement
DetailsA existing structure's (PDB:8UHW) coordinates were relaxed using the Rosetta Fast Relax algorithm and refined against the EM density from cryoSPARC using Phenix and Coot. Histidine residues coordinate the iron ion; short distances flagged as clashes correspond to metal coordination geometry.
RefinementProtocol: RIGID BODY FIT
Output model

PDB-13he:
CryoEM structure of AdhE spirosome from Clostridium thermocellum uncovered by visual proteomics.

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