Time-resolved Cryo-EM Jack Fu Joachim Franks lab Columbia - - PowerPoint PPT Presentation

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Time-resolved Cryo-EM Jack Fu Joachim Franks lab Columbia - - PowerPoint PPT Presentation

Jun 17, 2023 156 likes •548 views

Time-resolved Cryo-EM Jack Fu Joachim Franks lab Columbia University Questions to address? How can time-resolved cryo-EM help you in your research? We need your help. What are the obstacles to success? There are a lot of

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Time-resolved Cryo-EM Jack Fu Joachim Frank’s lab Columbia University

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Questions to address?

  • How can time-resolved cryo-EM help you in your research?
  • We need your help.
  • What are the obstacles to success?
  • There are a lot of issues in time-resolved Cryo-EM method.
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Time-resolved cryo-electron microscopy

  • Time-resolved cryo-electron microscopy (cryo-EM) combines the

known advantages of single-particle cryo-EM in visualizing molecular structure with the ability to dissect the time progress of a reaction between molecules in vitro.

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Time-resolved cryo-electron microscopy

  • Time-resolved cryo-electron microscopy (cryo-EM) combines the

known advantages of single-particle cryo-EM in visualizing molecular structure with the ability to dissect the time progress of a reaction between molecules in vitro.

Molecule A + Molecule B Molecule C Intermediate 1 (lifetime: min, sec, ms, ms or even shorter) Light, pH, Ionic concentration, Temperature, Electric Field, Magnetic Field, Mechanical force, Others.

?

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What has been tested?

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Acetylcholine receptor Acetylcholine

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Limitations in the spraying-freezing method

Molecule A + Molecule B On Cryo-EM grid Small molecule Acetylcholine/ ATP Marker to identify the droplets +

The mixing is dependent on diffusion

  • 1. slow
  • 2. dependence on molecular weight

Our solution is the mixing-spraying-freezing method

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Experimental setup – Microfluidic chip

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Experimental setup – Mixer performance

Not mixed well Flow rate: 1mL/s Very well mixed Flow rate: 6 mL/s

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Experimental setup – Mixer performance

Mixing time 0.5 ms Mixing 70S ribosomes and Ferritin molecules

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Experimental setup – Reaction time

Length of the reaction channels and the flow rate determine the reaction time in the microfluidic chips.

Mixing time 0.5 ms Reaction time 4-500 ms

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Experimental setup – Plunging and freezing

Reaction is stopped by plunging into cryogen.

Cryogen Mixing time 0.5 ms Reaction time 4-500 ms Plunging time 18 ms

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Limitation

  • 1. How to get right ice thickness?
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Blotting grid Spraying grid

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Quantifoil R1.2/1.3 400 mesh

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Blotting grid Spraying grid

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Spraying grid

Grid Bar Grid Bar Holey carbon film droplet

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Spraying grid

Grid Bar Grid Bar Holey carbon film droplet

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Spraying grid

Grid Bar Grid Bar Holey carbon film droplet

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(A and B) The ice thickness is different from the leading to the trailing side of each hole (blue arrows), which is different from grids obtained by the blotting method. (C and D) The ice is thinner on one side than on the other side as indicated by the different lengths of the tunnels drilled on the two sides. The thicker ice

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Data collection

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Mean droplet size – flow rate ratio between liquid and gas

  • Diameter: 36.2 to 4.4 μm ( Volume : 24.4 pL - 0.044 pL )

Where m is the mass flow rate, and subscripts g and l denote gas and liquid. Suppose that the solution sprayed is water, the viscosity μ, surface tension σ, density ρ are 0.89 × 10−3 Pa·s, 0.072 N/m, 1 × 103 kg/m3, respectively.

Gas pressure: 16 psi to 48 psi

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Measurements of Ice Thickness of Droplets Sprayed on the EM Grid

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Pie charts illustrating the droplet siz ize dis istribution under four dif ifferent sprayin ing conditio ions.

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3.0-Å Resolution Structure of Apoferritin Obtained by Spraying with the Microsprayer

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360-kDa membrane protein, AcrB 3.7 Å

8.0 Å 5.5 Å 3.0 Å

In preparation

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3D EM-density (3.2 Å) of AcrB in Native Cell Membrane Nanoparticle

Submitted

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3D EM-density: Native Cell Membrane Bilayer

PE

Submitted

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Lipid Belt in Sliced View and Hexagonal Pattern of Lipid Arrangement

Submitted

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TM Schmeing & V Ramakrishnan Nature 000, 1-9 (2009) doi:10.1038/nature08403

Overview of translation

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The recycling process

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Next steps

  • 1. General application
  • 2. Nano-fluidic system (less sample consumption)
  • 3. Sub-millisecond system
  • (mixing time < 50 ms, freezing time < 100 ms)
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Energy filter 20 eV (red) vs no slit (blue)

Low frequency SSNR (20-200 A) High frequency SSNR (4-10 A)

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3D sprayer Microfluidic device with PDMS

  • Dept. of Mechanical Engineering

Columbia University

  • Dr. Qiao Lin

Xiangsong Feng Yuan Jia

  • Prof. Howard D. White

Eastern Virginia Medical School

New Time-Resolved Machine Frank nk Lab Tea eam

  • Prof. Joachim Frank

Sandip Kaledhonkar Bo Chen Ming Sun Mans Anneli Ruben Kelvin

Gonzalez Lab Ehren enberg Lab ab

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References

Key intermediates in ribosome recycling visualized by time-resolved cryoelectron microscopy Z Fu, S Kaledhonkar, A Borg, M Sun, B Chen… - Structure, 2016 A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM X Feng*, Z Fu*, S Kaledhonkar, Y Jia, B Shah, A Jin… - Structure, 2017 Lipid Bilayer Structure in Native Cell Membrane Nanoparticles of Multidrug Exporter AcrB Qiu W*, Z Fu*, Xu G, … - Submitted