|Entry||Database: PDB / ID: 7vdc|
|Title||3.28 A structure of the rabbit muscle aldolase|
|Components||Fructose-bisphosphate aldolase A|
|Keywords||STRUCTURAL PROTEIN / Rabbit muscle aldolase|
|Function / homology|
Function and homology information
negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I band / glycolytic process / protein homotetramerization / positive regulation of cell migration
Similarity search - Function
Fructose-bisphosphate aldolase class-I active site. / Fructose-bisphosphate aldolase class-I active site / Fructose-bisphosphate aldolase, class-I / Fructose-bisphosphate aldolase class-I / Aldolase-type TIM barrel
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A
Similarity search - Component
|Biological species||Oryctolagus cuniculus (rabbit)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å|
|Authors||Fan, H.C. / Zhang, Y. / Sun, F.|
|Funding support|| China, 5items |
|Citation||Journal: Nat Commun / Year: 2021|
Title: A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI.
Authors: Hongcheng Fan / Bo Wang / Yan Zhang / Yun Zhu / Bo Song / Haijin Xu / Yujia Zhai / Mingqiang Qiao / Fei Sun /
Abstract: Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, ...Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
|Structure viewer||Molecule: |
Downloads & links
A: Fructose-bisphosphate aldolase A
B: Fructose-bisphosphate aldolase A
C: Fructose-bisphosphate aldolase A
D: Fructose-bisphosphate aldolase A
Mass: 39394.875 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P00883, fructose-bisphosphate aldolase
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Rabbit muscle aldolase / Type: COMPLEX / Entity ID: all / Source: NATURAL|
|Molecular weight||Value: 0.15 MDa / Experimental value: NO|
|Source (natural)||Organism: Oryctolagus cuniculus (rabbit)|
|Source (recombinant)||Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)|
|Buffer solution||pH: 7.5|
|Specimen||Conc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: NICKEL/TITANIUM / Grid mesh size: 300 divisions/in.|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 215000 X|
|Image recording||Electron dose: 70 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 2460|
|Image scans||Movie frames/image: 40|
|Software||Name: PHENIX / Version: 1.19_4092: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Num. of particles selected: 769925|
|Symmetry||Point symmetry: D2 (2x2 fold dihedral)|
|3D reconstruction||Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 228832 / Algorithm: FOURIER SPACE / Symmetry type: POINT|
|Refine LS restraints|
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