A Crash Course in Cryo-EM Sample Preparation
MARK HERZIK, Ph.D. Assistant Professor University of California, San Diego Department of Chemistry and Biochemistry herziklab.com @mherzik ( ) 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 Every Specimen Requires Careful Optimization It’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 Every Specimen Requires Careful Optimization It’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 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-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-9599 EMD-9865 EMD-9914 EMD-10012 EMD-10101 EMD-10205 EMD-10533 EMD-10675 EMD-10712 EMD-10714
EMD-11103 EMD-11121 EMD-11122 EMD-20026 EMD-20027 EMD-20028 EMD-20112 EMD-20155 EMD-20156 EMD-20157 EMD-20521 EMD-20837
EMD-21024 EMD-21951 EMD-22346 EMD-22347 EMD-22348 EMD-22349 EMD-22350 EMD-22351 EMD-30083 EMD-30084 Numerous Approaches For Optimizing Each Specimen 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-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-9599 EMD-9865 EMD-9914 EMD-10012 EMD-10101 EMD-10205 EMD-10533 EMD-10675 EMD-10712 EMD-10714 Structures that utilized “traditional” grid supports and blotters
EMD-11103 EMD-11121 EMD-11122 EMD-20026 EMD-20027 EMD-20028 EMD-20112 EMD-20155 EMD-20156 EMD-20157 EMD-20521 EMD-20837
EMD-21024 EMD-21951 EMD-22346 EMD-22347 EMD-22348 EMD-22349 EMD-22350 EMD-22351 EMD-30083 EMD-30084 Numerous Approaches For Optimizing Each Specimen 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-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-9599 EMD-9865 EMD-9914 EMD-10012 EMD-10101 EMD-10205 EMD-10533 EMD-10675 EMD-10712 EMD-10714 Structures that utilized next generation grid supports with traditional blotters
EMD-11103 EMD-11121 EMD-11122 EMD-20026 EMD-20027 EMD-20028 EMD-20112 EMD-20155 EMD-20156 EMD-20157 EMD-20521 EMD-20837
EMD-21024 EMD-21951 EMD-22346 EMD-22347 EMD-22348 EMD-22349 EMD-22350 EMD-22351 EMD-30083 EMD-30084 Numerous Approaches For Optimizing Each Specimen 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-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-9599 EMD-9865 EMD-9914 EMD-10012 EMD-10101 EMD-10205 EMD-10533 EMD-10675 EMD-10712 EMD-10714 Structures that utilized solid support layers
EMD-11103 EMD-11121 EMD-11122 EMD-20026 EMD-20027 EMD-20028 EMD-20112 EMD-20155 EMD-20156 EMD-20157 EMD-20521 EMD-20837
EMD-21024 EMD-21951 EMD-22346 EMD-22347 EMD-22348 EMD-22349 EMD-22350 EMD-22351 EMD-30083 EMD-30084 Numerous Approaches For Optimizing Each Specimen 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-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-9599 EMD-9865 EMD-9914 EMD-10012 EMD-10101 EMD-10205 EMD-10533 EMD-10675 EMD-10712 EMD-10714 Structures that utilized next generation sample application technologies
EMD-11103 EMD-11121 EMD-11122 EMD-20026 EMD-20027 EMD-20028 EMD-20112 EMD-20155 EMD-20156 EMD-20157 EMD-20521 EMD-20837
EMD-21024 EMD-21951 EMD-22346 EMD-22347 EMD-22348 EMD-22349 EMD-22350 EMD-22351 EMD-30083 EMD-30084 Biological Specimen Preparation for Single-Particle CryoEM Basic workflow - each step can inform on the others
Sample Purification & EM Image Acquisition & EM Sample Preparation Optimization Screening Principles Of Thin-Film Vitrification & Plunge Freezing Biological 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
Silicon Oxide
Images adapted from Matthijn Vos - Cold Spring Harbor Lab Cryo-EM Course Principles Of Thin-Film Vitrification & Plunge Freezing Biological 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 Slow Freezing Sample Warming
Silicon Oxide Amorphous, vitreous ice Hexagonal Ice Cubic Ice
Images adapted from Matthijn Vos - Cold Spring Harbor Lab Cryo-EM Course Principles Of Thin-Film Vitrification & Plunge Freezing Biological 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 • 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 History First EM specimens were vitrified in early 1980’s
Marc Adrian
Jean Lepault
Alasdair McDowall Jaques Dubochet Specimen Vitrification for Cryo-EM - A Brief History First EM specimens were vitrified in early 1980’s
J. Dubochet, J. Lepault, R. Freeman, J.A. Berriman, J.C. Homo. Electron microscopy of frozen water and aqueous solutions. Journal of Microscopy (1982) Specimen Vitrification for Cryo-EM - A Brief History First EM specimens were vitrified in early 1980’s
“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 Freezing How do we generate thin-films for vitrification?
Sgro &Costa, Front. Mol. Biosci., 31 July 2018 | https://doi.org/10.3389/fmolb.2018.00074 Principles of Thin-Film Vitrification & Plunge Freezing How do we generate thin-films for vitrification? 1984 2020
Sgro &Costa, Front. Mol. Biosci., 31 July 2018 | https://doi.org/10.3389/fmolb.2018.00074 Principles of Thin-Film Vitrification & Plunge Freezing How do we generate thin-films for vitrification?
4º C cold room
Postdoc Mark humidifier to increase humidity (>90%)
Sgro &Costa, Front. Mol. Biosci., 31 July 2018 | https://doi.org/10.3389/fmolb.2018.00074 Principles of Thin-Film Vitrification & Plunge Freezing How do we generate thin-films for vitrification?
Blotting paper Liquid Nitrogen Sample
Liquid Ethane
Sgro &Costa, Front. Mol. Biosci., 31 July 2018 | https://doi.org/10.3389/fmolb.2018.00074 Principles of Thin-Film Vitrification & Plunge Freezing How do we generate thin-films for vitrification?
Sgro &Costa, Front. Mol. Biosci., 31 July 2018 | https://doi.org/10.3389/fmolb.2018.00074 Developments In Automated Blotters >40 years later and most sample plungers still use Whatman Filter Paper #1…
Manual FEI Vitrobot Gatan Cryoplunge Leica EM GP What Holds Our Specimens? It all starts with a ~3mm grid…
protochips.com What Holds Our Specimens? It all starts with a ~3mm grid…
protochips.com
• Hole diameter: range of sizes from 0.6 µm to 8 µm • Hole spacing: range of sizes from 1 µm and 4 µm • Film material: Carbon, Gold Alloy • Grid material: Copper, Gold • Grid mesh size: 200, 300, 400 • Film thickness: 20 to 40 nm
https://scienceservices.de/en/tools-supplies/tem-grids/info-tem-grids/coatings-and-coating-materials What Holds Our Specimens? It all starts with a ~3mm grid…
~1 µm
protochips.com ~2.5 µm
• Hole diameter: range of sizes from 0.6 µm to 8 µm • Hole spacing: range of sizes from 1 µm and 4 µm • Film material: Carbon, Gold Alloy • Grid material: Copper, Gold • Grid mesh size: 200, 300, 400 • Film thickness: 20 to 40 nm
https://scienceservices.de/en/tools-supplies/tem-grids/info-tem-grids/coatings-and-coating-materials Desired Properties Of A Cryo-EM Grid I 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 films New materials afford improved stability
Glaeser & Henderson 2011 Grid Preparation Commercially 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 foil Gatan Solarus II Plasma Cleaner Behavior Of Particles In Open Holes Long 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
Noble et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife 2018 Behavior Of Particles In Open Holes
Noble et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife 2018 Behavior Of Particles In Open Holes The air-water interface: a massive hydrophobic effect
In a 10µm thick curtain of solution, EVERY protein will interact with air-water interface
This dramatically changes the energy landscape for denaturation
Trurnit J. Colloid Sci. 1960 Glaeser & Han. Biophys Rep 2017 Behavior Of Particles In Open Holes
Noble et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife 2018 Approaches To Overcome Air-Water Interface Issues Air-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) Behavior Of Particles In Open Holes
Noble et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife 2018 Approaches To Overcome Air-Water Interface Issues Air-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 (<200 kDa) proteins • Graphene and graphene oxide support films • Optically more transparent than amorphous carbon • Surface can be functionalized Grids For Cryo-EM - Solid Support Layers Avoiding the air-water interface
Film Materials: Amorphous Carbon Graphene Oxide Graphene Streptavidin monolayer
• 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 Russo & Passmore. Curr. Opinion Struct. Bio. 2016 Grids For Cryo-EM - Solid Support Layers Avoiding the air-water interface
Pantelic et al. J. Struct. Biol. 2009
Any film will add some background noise to images
Russo & Passmore. Curr. Opinion Struct. Bio. 2016 Grids For Cryo-EM - Solid Support Layers Avoiding the air-water interface
Watch out for grids with dry support films! 2/2/20 Watch out for dry grids with support films!
Pantelic et al. J. Struct. Biol. 2009
Any film will add some background noise to images Russo and Passmore 2014 Nat Methods
Russo & Passmore. Curr. Opinion Struct. Bio. 2016
Confidential 42 Grids For Cryo-EM - Solid Support Layers Mass-producible, functionalized graphene supports
Multiple functionalizations of graphene can be patterned on to each grid
K. Naydenova, M.J. Peet, C.J. Russo. Multifunctional graphene supports for electron cryomicroscopy. PNAS 2019 Approaches To Overcome Air-Water Interface Issues Air-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 (<200 kDa) proteins • Graphene and graphene oxide support films • Optically more transparent than amorphous carbon • Surface can be functionalized • Next generation of spraying, rapid-freezing devices • No blotting paper needed • Decreases time for samples to interact with air-water interface • Potential for time-resolved studies Alternative Approaches To Cryo-EM Grid Prep pL dispensers and self-wicking grids - Spotiton (a.k.a SPT Labtech Chameleon) The Spotiton Project
New York Structural Biology Center
Bridget Carragher Clint Potter
use pL amount of sample Razinkov et al. A new method for vitrifying samples for cryoEM. J Struct Biol (2016) Alternative Approaches To Cryo-EM Grid Prep pL dispensers and self-wicking grids - Spotiton (a.k.a SPT Labtech Chameleon) The Spotiton Project
New York Structural Biology Center
Bridget Carragher Clint Potter 1 droplets (32 pl)
1000 droplets (32 nl) Alternative Approaches To Cryo-EM Grid Prep Numerous other sample dispensers in development and production
Vitrojet Rubinstein et al. Shake-it-off: A simple ultrasonic cryo-EM specimen preparation device Yong Zi Tan & John Rubinstein Acta Cryst D doi.org/10.1107/ Back-it-up! A simple ultrasonic S2059798319014372 cryo-EM specimen preparation device BioRxiv.org doi.org/ 10.1101/2020.05.03.075366 Biological Specimen Preparation for Single-Particle CryoEM Basic workflow - each step can inform on the others
Sample Purification & EM Image Acquisition & EM Sample Preparation Optimization Screening Importance Of Databases In Sample Screening Always 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 (<100 kDa) and flexible proteins by cryo-EM is not for the faint of heart
• A Krios will not magically solve all your sample preparation issues (no scope will)
• Even a dedicated TEM cannot overcome sample preparation issues Why Do I Prefer To Manually Blot All My Specimens?
Vitrobot Manual Plunger