A Crash Course in Cryo-EM Sample Preparation...Aug 11, 2020 · Gatan Solarus II Plasma Cleaner. Behavior Of Particles In Open Holes Long story short: less than ideal Noble et al

MARK HERZIK, Ph.D. Assistant Professor

University of California, San Diego Department of Chemistry and Biochemistry

herziklab.com @mherzik ( )

A Crash Course in Cryo-EM Sample Preparation

http://herziklab.com

What Prevents Us From Routinely Reaching Atomic Resolution In Cryo-EM of Biological Samples?

Hint: It’s not the optics of the microscope…

Nakane et al. 2020 BioRxiv.org

Yip et al. 2020 BioRxiv.org

http://BioRxiv.orghttp://BioRxiv.org

Every Specimen Requires Careful OptimizationIt’s critical to optimize each and every step specifically for your specimen

Very first screening dataset - 15Jan03 Final high-resolution dataset - 16Sep09

5 different constructs

8 different detergent/nanodisc

3 different grid types

>200 grids frozen and screened

5 high-resolution data collections

Numerous Approaches For Optimizing Each Specimen

EMD-0144 EMD-0263 EMD-2788 EMD-3853 EMD-3854 EMD-4213 EMD-4485 EMD-4698


EMD-4701 EMD-4905 EMD-6800 EMD-6801

EMD-6802 EMD-8428 EMD-10712 EMD-10714

EMD-11103 EMD-11121 EMD-11122 EMD-20026 EMD-20027 EMD-20028 EMD-20155 EMD-20156 EMD-20157 EMD-20521 EMD-20837

EMD-21024 EMD-22351 EMD-30083 EMD-30084EMD-21951 EMD-22346 EMD-22347 EMD-22348 EMD-22349 EMD-22350

EMD-20112

There are a lot of resources available - old and new - that can be used (and more on the way!)Cryo-EM’s version of hen egg white lysozyme - heavy chain apoferritin




EMD-4701 EMD-4905 EMD-6800 EMD-6801

EMD-6802 EMD-8428 EMD-10712 EMD-10714



Structures that utilized “traditional” grid supports and blotters

EMD-20112

There are a lot of resources available - old and new - that can be used (and more on the way!)Cryo-EM’s version of hen egg white lysozyme - heavy chain apoferritin




EMD-4701 EMD-4905 EMD-6800 EMD-6801

EMD-6802 EMD-8428 EMD-10712 EMD-10714



EMD-20112

There are a lot of resources available - old and new - that can be used (and more on the way!)

Structures that utilized next generation grid supports with traditional blotters

Cryo-EM’s version of hen egg white lysozyme - heavy chain apoferritin




EMD-4701 EMD-4905 EMD-6800 EMD-6801

EMD-6802 EMD-8428 EMD-10712 EMD-10714



EMD-20112


Structures that utilized solid support layers





EMD-4701 EMD-4905 EMD-6800 EMD-6801

EMD-6802 EMD-8428 EMD-10712 EMD-10714



EMD-20112


Structures that utilized next generation sample application technologies


Biological Specimen Preparation for Single-Particle CryoEMBasic workflow - each step can inform on the others

Sample Purification & Optimization EM Sample Preparation

EM Image Acquisition & Screening

Silicon Oxide

Principles Of Thin-Film Vitrification & Plunge FreezingBiological specimens do not like vacuums - need to freeze them!

The specimen must first be vitrified before imaging:• Vitrification:

• Transformation of water from a liquid to a solid amorphous state w/o nucleation of ice crystals • Nucleation of ice crystals is time, temperature, and pressure dependent

Glass (amorphous)

Crystals

Images adapted from Matthijn Vos - Cold Spring Harbor Lab Cryo-EM Course




Amorphous, vitreous iceSilicon Oxide Hexagonal Ice

Slow Freezing

Cubic Ice

Sample Warming

Images adapted from Matthijn Vos - Cold Spring Harbor Lab Cryo-EM Course




• Maintains the specimen in a “near-native” environment • Freeze the specimen sufficiently fast to prevent ice formation

• Renders the specimen suitable to imaging in a vacuum• Prevents dehydration

• Immobilizes specimen without requirement of fixation• At least until you illuminate it with an electron beam…

Specimen Vitrification for Cryo-EM - A Brief HistoryFirst EM specimens were vitrified in early 1980’s

Jaques Dubochet

Alasdair McDowall

Marc Adrian

Jean Lepault


J. Dubochet, J. Lepault, R. Freeman, J.A. Berriman, J.C. Homo. Electron microscopy of frozen water and aqueous solutions. Journal of Microscopy (1982)


“It came as a surprise when it was found that the easiest way to obtain a thin water film is to stretch it, without any support, over the holes of a grid … either by pipetting away excess water or removing it with blotting paper”

J. Dubochet, M. Adrian, J. Chang, J.C. Homo, J. Lepault, A. McDowall, P. Schultz. Cryo-electron microscopy of vitrified specimens. Quarterly Review of Biophysics (1988)

Principles of Thin-Film Vitrification & Plunge FreezingHow do we generate thin-films for vitrification?

Sgro &Costa, Front. Mol. Biosci., 31 July 2018 | https://doi.org/10.3389/fmolb.2018.00074

https://doi.org/10.3389/fmolb.2018.00074



20201984




4º C cold room

humidifier to increase humidity

(>90%)

Postdoc Mark




Liquid Ethane

Liquid Nitrogen

Blotting paper

Sample


Developments In Automated Blotters>40 years later and most sample plungers still use Whatman Filter Paper #1…

FEI Vitrobot Gatan Cryoplunge Leica EM GPManual

What Holds Our Specimens?It all starts with a ~3mm grid…

protochips.com

http://protochips.com


https://scienceservices.de/en/tools-supplies/tem-grids/info-tem-grids/coatings-and-coating-materials

protochips.com

• Hole diameter:• Hole spacing:

• Film material:• Grid material:

• Grid mesh size:• Film thickness:

range of sizes from 0.6 µm to 8 µm range of sizes from 1 µm and 4 µm Carbon, Gold Alloy Copper, Gold 200, 300, 400 20 to 40 nm



https://scienceservices.de/en/tools-supplies/tem-grids/info-tem-grids/coatings-and-coating-materials

protochips.com

• Hole diameter:• Hole spacing:

• Film material:• Grid material:

• Grid mesh size:• Film thickness:

range of sizes from 0.6 µm to 8 µm range of sizes from 1 µm and 4 µm Carbon, Gold Alloy Copper, Gold 200, 300, 400 20 to 40 nm

~1 µm

~2.5 µm


Desired Properties Of A Cryo-EM GridI would consider these the bare minimum

Grid supports should have the following properties:• Possess structural integrity

• Necessary to support the thin film encompassing our specimen

• Must be quite electron stable• Must withstand large electron doses without being compromised

• Inert*• Does not chemically modify or alter specimen

• Well-defined spatial relationship of the squares and holes to allow for the use of automated imaging techniques

More detail about support filmsNew materials afford improved stability

Glaeser & Henderson 2011

Grid PreparationCommercially purchased support films are usually hydrophobic

Emitech K950x Turbo Evaporator

• Glow discharging in a plasma cleaner for 5-30s • This increases the hydrophilicity of the grid - temporarily!

• Allows for better dispersion of liquid across foilGatan Solarus II Plasma Cleaner

Behavior Of Particles In Open HolesLong story short: less than ideal

Noble et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife 2018

Behavior Of Particles In Open Holes



Trurnit J. Colloid Sci. 1960

In a 10µm thick curtain of solution,EVERY protein will interact with air-water interface

This dramatically changes the energy landscape for denaturation

Glaeser & Han. Biophys Rep 2017

The air-water interface: a massive hydrophobic effect

Approaches To Overcome Air-Water Interface IssuesAir-water interface can’t be escaped (yet) but effects can be lessened

Modifications to grid preparation:• Addition of surfactants:

• Adhere to the air-water interface and limit protein interaction at surface • Amphipol A8-35, NP-40, n-dodecyl-ß-D maltose (DDM), lauryl maltose neopentyl glycol (LMNG)




• Amorphous carbon support films• Surface can be functionalized • Cheap and easy to make • Less than ideal for small (

Grids For Cryo-EM - Solid Support LayersAvoiding the air-water interface

• Provides surface for particles to adhere • Can be functionalized • Eliminates some of the problems with the air:water interface • Optically “transparent” in the electron microscope

Film Materials: Amorphous Carbon Graphene Oxide Graphene Streptavidin monolayer

Russo & Passmore. Curr. Opinion Struct. Bio. 2016



Pantelic et al. J. Struct. Biol. 2009

Any film will add some background noise to images



Pantelic et al. J. Struct. Biol. 2009

Any film will add some background noise to images

2/2/20

Confidential

42

Watch out for dry grids with support films!

Russo and Passmore 2014 Nat Methods

Watch out for grids with dry support films!

Grids For Cryo-EM - Solid Support LayersMass-producible, functionalized graphene supports

K. Naydenova, M.J. Peet, C.J. Russo. Multifunctional graphene supports for electron cryomicroscopy. PNAS 2019

Multiple functionalizations

of graphene can be

patterned on to each grid




• Amorphous carbon support films• Surface can be functionalized • Cheap and easy to make • Less than ideal for small (

Alternative Approaches To Cryo-EM Grid PreppL dispensers and self-wicking grids - Spotiton (a.k.a SPT Labtech Chameleon)

Bridget Carragher Clint Potter

New York Structural Biology Center

use pL amount of sample

The Spotiton Project

Razinkov et al. A new method for vitrifying samples for cryoEM. J Struct Biol (2016)

Alternative Approaches To Cryo-EM Grid PreppL dispensers and self-wicking grids - Spotiton (a.k.a SPT Labtech Chameleon)

Bridget Carragher Clint Potter

New York Structural Biology Center

The Spotiton Project

1000 droplets (32 nl)

1 droplets (32 pl)

Alternative Approaches To Cryo-EM Grid PrepNumerous other sample dispensers in development and production

Vitrojet Rubinstein et al. Shake-it-off: A simple ultrasonic cryo-EM specimen preparation device Acta Cryst D doi.org/10.1107/S2059798319014372

Yong Zi Tan & John Rubinstein Back-it-up! A simple ultrasonic cryo-EM specimen preparation device BioRxiv.org doi.org/10.1101/2020.05.03.075366

Biological Specimen Preparation for Single-Particle CryoEMBasic workflow - each step can inform on the others

Sample Purification & Optimization EM Sample Preparation

EM Image Acquisition & Screening

Importance Of Databases In Sample ScreeningAlways best to understand where your good images came from - and why

Some Important Last Remarks

• No two grids are ever the same, even when frozen back-to-back

• Screen, screen, screen and screen again before collecting tons of data

• Use as much data from sample screening to optimize sample and grid preparation

• Images for most specimens will not look like what you see in methods papers

• Working will small (

Why Do I Prefer To Manually Blot All My Specimens?

Vitrobot Manual Plunger

Documents

A Crash Course in Cryo-EM Sample Preparation...Aug 11, 2020 · Gatan Solarus II Plasma Cleaner. Behavior Of Particles In Open Holes Long story short: less than ideal Noble et al