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