Cryo-TEM sample preparation for molecular imaging · Cryo-TEM sample preparation for molecular...

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Qing Wang (王庆), Ph.D.

Cryo-TEM sample preparation for molecular imaging

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Why cryo-fixation/vitrification?

A very brief histry

Practical aspects of thin-film vitrification

Possible issues and our strategies


What prevents us from routinely achieving atomic resolution in life sciences?

1nm

0.135nm Si


Near-atomic resolution from Single-Particle-Analysis cryo-EM


The workflow of SPA cryo-TEM

When things work really well:

A few weeks

A few hours

3 to 5 days

A few weeks

The reality:

A few weeks or months

(A few hours) x N

A few days or weeks

A few weeks or months


Challenges/goals for biological specimens preparation

Preserve macromolecules in aqueous solutions at near-native state

• Water-rich environment vs high-vacuum

• Radiation damage

Improve image contrast and resolution

• Weak scatters of electrons

• Low contrast/density difference


Negative staining

Advantages

• Quickly screen lots of conditions • Very high contrast • Higher electron dose allowed, due to heavy metal shielding • Relatively easy Limitations

• Limited resolution (20 Å due to grain size) • Sample molecules may be damaged or distorted • Uneven staining, low reproducibility


Immobilized solution?

High-vacuum environment

Cooling to immobilize molecules and reduce evaporation rate?


Water is difficult to understand!

Liquid : • local tetrahedral symmetry • great mobility (exchange time 10-12s) • long list of models for structure

Ice: • Hexagonal ice • Cubic ice • >10 solid polymorphs (at high pressure) • Vitreous ice (amorphous or glassy ice)


Vitrification is the key!

• Vitrification:

- transformation of liquid water to an amorphous state

- avoiding the nucleation of ice crystals

• Ice expands:

- potential of breaking cells and proteins

• Ice produces contrast

- interfering with sample density


Early days of cryo-TEM sample preparation

Fernandez-Moran et al early advocator of cryo-TEM

1960s Inventor of diamond knife and cryo-ultramicrotome Parsons et al liquid hydration / Environmental TEM

1974 Atomic resolution of wet biological specimen preserved in TEM Taylor & Glaeser frozen hydration 1974 Frozen hydrated crystal of biological specimens Unwin & Henderson glucose embedding 1975 3D model of purple membrane at 7Å Dubochet et al 1984 Thin-film vitrification to observe frozen-hydrated biological specimens


Thin-film vitrification

Jacques Dubochet


Thin-film vitrification

• The nucleation of ice crystals depends on temperature, pressure, and freezing time (cooling rate);

• Cooling rate depends on the thermal properties of water, the sample thickness, and the heat extraction from specimen surface.

• Adsorption of a suspension to grids with/without carbon support film

• Blotting with filter paper to remove excess liquid formation of a very thin suspension film

• Plunging of the sample in a cryogen leads to

immediate freezing of the thin film formation of a vitreous thin layer of ice


“Robotic” sample handling

Controlled blotting parameter

Controlled humidity and temperature

Better reproducibility


Practical aspects of cryo-TEM sample preparation

Biological sample (size, amount, purity)

TEM grids (brand, metal, mesh size, support film)

Plasma cleaner

Prepare the cryogen

Plunge-freezing tool (settings and parameters)

Objectives


Biological samples: how small/big can my protein be?

• Can we tell what are and what are not our particles?

• Proteins smaller than 200 kD are often challenging;

• It really depends on what you want to achieve; • A few examples are given bellow:

Spliceosome 3.6 Å

64 kD hemoglobin 3.2 Å

300 kD TRPV1 3.4 Å


Biological samples: concentration, amount, and buffer

• 4 μl for each grid, at 1 to 3 mg/ml concentration. - 10x the concentration used for negative staining - expected particle distribution on holey grids

• Is my sample still stable at these conditions? - check with SEC - GraFix

Vinothkumar and Henderson (2016)


Biological samples: concentration, amount, and buffer

• Avoid glycerol, sucrose, and high concentration of salt. - Protein: 1.36 g/cm3

• Watch out for detergents


Biological samples: what purity does my sample need to be?

• Looks homogeneous on SDS-PAGE / native PAGE

• Characterization with biophysical techniques: - Mass Spectrometry (MW, PTMs) - DLS etc

• Have a quick look with negative staining or diagnostic cryo-EM

• Compositional vs conformational heterogeneity


Biological samples: what purity does my sample need to be?

Fernandez et al, (2013)

Making use of compositional heterogeneity: EM-guided sample preparation


What type of grids should I use?

Metal: Copper, Nickel, Gold, Molybdenum

Mesh size: Support vs usable area (e.g. 400 mesh)

Support film: holey carbon (+ thin carbon, graphene)

Hole size: 1 to 2 μm (R2/2)

GIG

shiny and dull side


Making the grid surface hydrophilic

Carbon films become progressively more hydrophobic over time. It is usually necessary to make them hydrophobic so the liquid sample can spread evenly over its surface. The procedure is called plasma cleaning/glow-discharging where plasma is created by ionization of specific gas(s) or air under low vacuum.

Hydrophobic surface

Hydrophilic surface


Prepare the cryogen

Cryogens Melting point (°C)

Boiling point (°C)

Relative cooling

efficiency* Ethane -183 -89 1.3

Liquid nitrogen -210 -196 0.1 Propane -189 -42 1.0 Freon 22 -160 -41 0.7

*relative to propane at 1.0

Liquid N2 -196°C

Liquid ethane (>99.9% purity)

-180°C


IMPORTANT: remarks on health and safety

• Cryogens (LN2) have a large liquid to gas expansion ratio and therefore it is important to work in a well-ventilated area to avoid potential suffocation.

• Cryogens (liquid ethane) can cause extensive burns. Wear gloves, goggles, or a mask.

• Ethane is an explosive gas and all steps should be performed in an explosion proof fume-hood with no possible source of ignition present.


Plunge-freezing tool

Home-made guillotine Vitrobot

Temperature drop at 105-106 K/s (Dubochet et al, 1988) - A cryogen with high

thermal conductivity

- Thin sample (<1 µm) as water is a poor thermal conductor

- Plunge at >1 m/s for convective heat transfer


Vitrobot: the blotting procedure

EM tweezers

Filter paper disks

Sample on grid

- Blotting force

- Blotting time

- Temperature

- Relative humidity

• Adsorption of a suspension to a grid with support film

• Plunging of the sample in a cryogen leads to immediate freezing of the thin film Formation of an amorphous (vitreous) thin layer of ice

• Blotting of excess liquid formation of a very thin suspension film (~100nm)


Vitrobot: the blotting procedure

• Air-water interface

• Water-carbon interface

• Water-metal interface


Controlled blotting conditions: temperature and relative humidity

• Evaporation would cause the solutes to concentrate, affect air/liquid interface, and how macromolecules organize around the surface;

• It is important to have high relative humidity (>80%) in the immediate area of the sample area.

From: Cryoelectron Microscopy of Liposomes; Peter M. Frederik and D. H. W. Hubert


Objectives of cryo-TEM sample preparation

• Varies depending the stage of the research project

• Stepwise approach may be a good practice

• How good is good enough?

3 day data collection session 60 hr x 50 images /hr = 3000 images 2 images/hole x 100 holes/square = 200 images/grid square we need 15 grid squares, i.e. <10% of one single grid


Material and methods used for sample preparation

Biological sample (size, amount, purity)

TEM grids (brand, metal, mesh size, support film)

Plasma cleaner

Prepare the cryogen

Plunge-freezing tool (settings and parameters)

Objectives


Hexagonal ice

Brag reflections

“Too slow freezing” Possible causes: cryogen temperature high mass


Cubic ice

“Partial warming up” Possible causes: specimen in touch with warm objects (>-130°C)


Cryo-TEM sample transfer and loading

Cryo-holder and cryo-transfer Autoloader and Cassette

Anti-contaminator


More bad ice

20e/Å2 - 80e/Å2

Transfer ice (from atmosphere) Vacuum contamination

Leopard skin Transfer ice and more


Broken squares often cause mechanic instability

Cryo-crinkling

Metal shrinks a lot Carbon shrinks less Ice expands


• Clean and vitreous ice, with minimal contamination

• Intact and flat ice across the grid square • Ice thickness

• Particle concentration and distribution


Ice thickness across the grid and different holes

Thickness gradient across the grid

~1 µm

~2.5 µm

Iempty Iice

Icarbon

good

bad


Thin ice, but not too thin

Hole center too thin Freeze dried?

Thicker area: well distributed particles

Thinner area: higher contrast but preferred orientation

Partially dehydrated


Particle distribution (concentration and orientation)

• Better distribution higher throughput

• Too many particles per micrograph - ice thickness - protein aggregates (surface chemistry) • Too few or no particles - ice thickness - sticking to carbon area?

change glow-discharge conditions change buffer conditions multiple rounds of sample application and blotting add a thin (4 nm) layer of continuous carbon film or graphene


Use graphene to control protein adsorption

Russo & Passmore Sader & Rosenthal Stahlberg et al


Particle distribution (concentration and orientation) - continued

• Particles with preferred orientation - ice thickness - battle against the air-water interface


Particle distribution (concentration and orientation) - continued

• Particles with preferred orientation - ice thickness - battle against the air-water interface (leading to protein denature)

add a thin (4 nm) layer of continuous carbon film or graphene plasma cleaning condition (add amylamine etc) change buffer conditions protein surface chemistry stick something to the target molecule

affinity grids (antibody, tagging systems)

tilting during data acquisition (Tan et al, 2017)

3D DNA origami as a scaffold


Take-home messages and questions

• The thin-film vitrification procedure offers a simple method for cryo-TEM sample preparation;

• It often takes time before one can get clean, thin

vitreous ice with good particle distribution;

• This is currently one major bottleneck for cryo-TEM studies.


Take-home messages and questions

• Concerted efforts to move from “trial-and-error art” to “systematic prediction, screening and optimization”?

• Are there alternatives to the thin-film plunge-freezing

method?

Picolitre liquid dispenser + self-blotting girds Carragher et al

Tears of wine Marangoni effect Glaeser et al

Cryo-TEM sample preparation for molecular imaging · Cryo-TEM sample preparation for molecular...

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