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- PDB-8s7h: Fructose 6-phosphate aldolase (FSA) from Escherichia coli -

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Basic information

Entry
Database: PDB / ID: 8s7h
TitleFructose 6-phosphate aldolase (FSA) from Escherichia coli
ComponentsFructose-6-phosphate aldolase 1
KeywordsLYASE / fructose 6-phosphate aldolase
Function / homology
Function and homology information


ketone catabolic process / fructose 6-phosphate aldolase activity / Lyases; Carbon-carbon lyases; Aldehyde-lyases / fructose metabolic process / identical protein binding / cytoplasm
Similarity search - Function
Fructose-6-phosphate aldolase / Transaldolase type 3B/Fructose-6-phosphate aldolase / Transaldolase/Fructose-6-phosphate aldolase, archaeal/bacterial / Transaldolase active site. / Transaldolase, active site / Transaldolase signature 1. / Transaldolase/Fructose-6-phosphate aldolase / Transaldolase/Fructose-6-phosphate aldolase / Aldolase-type TIM barrel
Similarity search - Domain/homology
Fructose-6-phosphate aldolase 1
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHebert, H. / Widersten, M.
Funding support1items
OrganizationGrant numberCountry
Other private218-0061
CitationJournal: Structure / Year: 2024
Title: Structure of the iminium reaction intermediate in an engineered aldolase explains the carboligation activity toward arylated ketones and aldehydes.
Authors: Hans Hebert / Eda Sönmez / Pasi Purhonen / Mikael Widersten /
Abstract: Two structures of fructose 6-phosphate aldolase, the wild-type and an engineered variant containing five active-site mutations, have been solved by cryoelectron microscopy (cryo-EM). The engineered ...Two structures of fructose 6-phosphate aldolase, the wild-type and an engineered variant containing five active-site mutations, have been solved by cryoelectron microscopy (cryo-EM). The engineered variant affords production of aldols from aryl substituted ketones and aldehydes. This structure was solved to a resolution of 3.1 Å and contains the critical iminium reaction intermediate trapped in the active site. This provides new information that rationalizes the acquired substrate scope and aids in formulating hypotheses of the chemical mechanism. A Tyr residue (Y131) is positioned for a role as catalytic acid/base during the aldol reaction and the different structures demonstrate mobility of this amino acid residue. Further engineering of this fructose 6-phosphate aldolase (FSA) variant, guided by this new structure, identified additional FSA variants that display improved carboligation activities with 2-hydroxyacetophenone and phenylacetaldehyde.
History
DepositionMar 1, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Fructose-6-phosphate aldolase 1
B: Fructose-6-phosphate aldolase 1
C: Fructose-6-phosphate aldolase 1
D: Fructose-6-phosphate aldolase 1
E: Fructose-6-phosphate aldolase 1
F: Fructose-6-phosphate aldolase 1
G: Fructose-6-phosphate aldolase 1
H: Fructose-6-phosphate aldolase 1
I: Fructose-6-phosphate aldolase 1
J: Fructose-6-phosphate aldolase 1


Theoretical massNumber of molelcules
Total (without water)238,95710
Polymers238,95710
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36110 Å2
ΔGint-326 kcal/mol
Surface area75580 Å2
MethodPISA

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Components

#1: Protein
Fructose-6-phosphate aldolase 1 / Fructose-6-phosphate aldolase A / FSAA


Mass: 23895.672 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Details: The C-terminal TSHHHHH is a His-tag not observed in the map.
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: fsaA, fsa, mipB, ybiZ, b0825, JW5109 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P78055, Lyases; Carbon-carbon lyases; Aldehyde-lyases
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: Fructose 6-phosphate aldolase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-AI
Buffer solutionpH: 8.35
Buffer componentConc.: 40 mM / Name: bicine / Formula: C6H13NO4
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 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 magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 48.64 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9816
Details: Images were collected in movie-mode, 45 frames at a dose of 1.08 e-/A2.
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1Xmippparticle selection
2EPU2.11.1image acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 251491
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162766 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 190 / Protocol: FLEXIBLE FIT / Space: REAL / Details: Initial fitting using Chimera and then Phenix
Atomic model buildingPDB-ID: 1L6W

1l6w


Accession code: 1L6W / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416380
ELECTRON MICROSCOPYf_angle_d0.97122290
ELECTRON MICROSCOPYf_dihedral_angle_d5.0352310
ELECTRON MICROSCOPYf_chiral_restr0.0472730
ELECTRON MICROSCOPYf_plane_restr0.0052860

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