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- EMDB-19773: Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-19773
TitleFructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant
Map dataFructose 6-phosphate aldolase, V134G129G166C107C163 mutant
Sample
  • Complex: Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant
    • Protein or peptide: Fructose-6-phosphate aldolase 1
  • Ligand: water
Keywordsfructose 6-phosphate aldolase / mutant / carboligation / LYASE
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 BL21 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHebert H / Widersten M
Funding support Sweden, 1 items
OrganizationGrant numberCountry
Other private218-0061 Sweden
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, 2024-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateMar 25, 2026-
Current statusMar 25, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19773.png

Downloads & links

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Map

FileDownload / File: emd_19773.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFructose 6-phosphate aldolase, V134G129G166C107C163 mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 180 pix.
= 147.6 Å		0.82 Å/pix.
x 180 pix.
= 147.6 Å		0.82 Å/pix.
x 180 pix.
= 147.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.08513269 - 0.15205924
Average (Standard dev.)0.0008227345 (±0.009987121)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 147.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Fructose 6-phosphate aldolase, V134G129G166C107C163 mutant, half1

Fileemd_19773_half_map_1.map
AnnotationFructose 6-phosphate aldolase, V134G129G166C107C163 mutant, half1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Fructose 6-phosphate aldolase, V134G129G166C107C163 mutant, half2

Fileemd_19773_half_map_2.map
AnnotationFructose 6-phosphate aldolase, V134G129G166C107C163 mutant, half2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant

EntireName: Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant
Components
  • Complex: Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant
    • Protein or peptide: Fructose-6-phosphate aldolase 1
  • Ligand: water

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Supramolecule #1: Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant

SupramoleculeName: Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli BL21 (bacteria)

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Macromolecule #1: Fructose-6-phosphate aldolase 1

MacromoleculeName: Fructose-6-phosphate aldolase 1 / type: protein_or_peptide / ID: 1
Details: The TSHHHHH is a C-terminal His-tag not observed in the map
Number of copies: 10 / Enantiomer: LEVO / EC number: Lyases; Carbon-carbon lyases; Aldehyde-lyases
Source (natural)Organism: Escherichia coli BL21 (bacteria)
Molecular weightTheoretical: 23.89065 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MELYLDTSDV VAVKALSRIF PLAGVTTNPS IIAAGKKPLD VVLPQLHEAM GGQGRLFAQV MATTAEGMVN DALKLRSIIA DIVV(A1H5N)VPVT AEGLAAIKML KAEGIPTCGT AVYGAAQGLL SALAGAEYVG PYVNVIDAQG GSGIQTVTDL HQLLK MHAP ...String:
MELYLDTSDV VAVKALSRIF PLAGVTTNPS IIAAGKKPLD VVLPQLHEAM GGQGRLFAQV MATTAEGMVN DALKLRSIIA DIVV(A1H5N)VPVT AEGLAAIKML KAEGIPTCGT AVYGAAQGLL SALAGAEYVG PYVNVIDAQG GSGIQTVTDL HQLLK MHAP QAKVCAAGFK TPRQALDCLL AGCESITLPL DVAQQMISYP AVDAAVAKFE QDWQGAFGRT SITSHHHHH

UniProtKB: Fructose-6-phosphate aldolase 1

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 272 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration2.0 mg/mL
BufferpH: 8 / Component - Concentration: 40.0 mM / Component - Formula: C6H13NO4 / Component - Name: bicine
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 8165 / Average exposure time: 2.2 sec. / Average electron dose: 50.04 e/Å2
Details: Images were collected in movie-mode, 40 frames with 1.251 e-/A2 dose/frame
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: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
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: 307612
CTF correctionSoftware - Name: CTFFIND / Type: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 115055
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationNumber classes: 3 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3wnq


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting was done using Chimera and Phenix was used for flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 156
Output model

PDB-8s7i:
Fructose 6-phosphate aldolase, L107C/A129G/R134V/L163C/S166G mutant

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