Improving every image:®
HexAuFoil ultra-small hole sample
supports for CryoEM reconstructions

ABSTRACTOver the last decade, cryo-electron microscopy (cryoEM) has undergone a “resolutionrevolution” and become a significant source of high-quality 3D-structural information forthe biological community. In fact, for some important drug targets, such as GPCRs andchannels, it has become the primary source of near-atomic resolution macromolecular
structural data. The resulting increasing use of cryoEM in drug discovery andmechanistic studies has driven a demand for ever higher quality and higher resolutiondata.
One continuing major source of information loss, and therefore map quality reduction,is particle movement during imaging. While data processing can reduce the effect of
this issue, novel sample preparation techniques are required to eliminate it. In thispresentation, we are excited to describe one such novel sample preparation device,developed at MRC Laboratory of Molecular Biology, Cambridge [1, 2], and currently
being commercialised by Quantifoil, part of SPT Labtech. HexAuFoil® ultra-small holegold supports reduce particle movement to sub-Ångstrom levels: the grids small holesize provides extra support to the thin ice layer that supports biological specimens
and thus reduces or eliminates ice buckling caused by the stress of cryo-cooling.
The resultant improvement in particle localisation enables maps to be extrapolated
to zero electron loss, before radiation damage. At the same time as reducing particle
movement, consideration has been given to increasing automated cryoEM throughput
at no cost to map quality with features such as maximal hole density.
Taken together, these developments mean that HexAuFoil® sample supports will
improve every image.
References[1] K Naydenova et al. Science 370: 223-226 (2020)[2] K Naydenova and C J Russo. Ultramicroscopy [online] 232: 113396 (2022)

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INTRODUCTION
WHY DO WE NEED A NEW GRID?

Over the last decade, the use of cryo-EM to study biomolecules has increasedexponentially, and it can now produce truly atomic resolution structures. However,sample preparation is widely recognised as a significant problem slowing the routine
use of the technique for drug discovery and basic research. Plunge-freezing of
biological samples is required for cryo-EM analysis, but results in the build up ofcompressive strain in the ice. Release of this strain due to electron-beam induced local
heating causes the image to blur, and reducing the quality and achievevable resolution.

SO WHAT ARE HEXAUFOIL AND WHO DEVELOPED
THEM?

Chris Russo and Katerina Naydenova (pictured below) of the MRC Laboratory ofMolecular Biology (LMB) in Cambridge, UK, created HexAuFoil® gold supports toovercome this issue. The <1 µm holes in the supports maintain a ratio of hole size toice thickness that prevents strain building to a high enough level for these effects tooccur. As a result, particles remain stationary and structures can be extrapolated tozero dose before the biomolecules are damaged by exposure to the electron beam. InChris Russo’s own words, “By using these grids, your data is always better: bringing
the movement to zero improves every image you take.”
An additional benefit is that the hexagonal hole arrangement has a high-packing
density that leads to higher data collection rates and therefore increases thethroughput of resolved structures which is important for rapid lead optimization.

3

WHAT IS A HEXAUFOIL® GRID?
HexAuFoil® sample supports consistof a holey gold foil on a hexagonal
gold mesh grid. The grid hexagons areapproximately 50 µm in size with ~1012 µm diameter bars. The gold foil
has 290 nm diameter holes arranged
in a hexagonal array with 310 nm
spacing, resulting in a 600 nm repeat,
as illustrated on the left.

50 μm

13 μm

290 nm
310 nm

4600 nmWHAT DO HEXAUFOIL® GRIDS LOOK LIKE?
UNDER A LIGHT MICROSCOPE
When inspected by eye, HexAuFoil® sample supports should appear a shiny gold,
similar to UltrAuFoil® grids. However, when using a microscope to check for foil
integrity, the appearance will change depending on the type of illumination selected.
Under reflected light, the grid bar side will be light yellow, and the foil side will beshiny with a brown tint (LHS in left image below). In contrast, if HexAuFoil® are viewed
using transmitted light, plasmon resonance from the regularly-arranged small holes
will make the foil appear blue (RHS in left image below). The same effect can makeparts of the foil in contact with grid appear green-blue in reflection (RHS in right imagebelow).
REFLECTED
LIGHT

TRANSMITTED
LIGHT

GRID
SIDE

FILM
SIDE

WHAT FEATURES DO THEY HAVE?
The grids include a number of fiducial marks to aid with orientation. There are 2 fiducialmarks on the rim of the grid which are visible by the naked eye (differing slightly fromthose illustrated here). In addition, there are a further 6 on grid fiducial marks to aidwith orientation. The largest on grid mark, bottom left in the illustration, is free of foil tofacilitate microscope alignments and flux measurements.
FIDUCIAL VISIBLE BY EYEADDITIONAL FIDUCIAL TO AIDORIENTATION IN MICROSCOPE
FOIL-FREE FIDUCIAL FOR
SCOP ADJUSTMENT AND FLUXMEASUREMENT

5DO YOU NEED TO MAKE CHANGES TO
YOUR SAMPLE PREPARATION PROTOCOL?

YES!

1. PLASMA CLEANINGHexAuFoil® grids should be plasma cleaned or glow discharged to increase theirwettability prior to use. In general, for best results, they will need glow discharging
for significantly longer than other sample supports, such as UltrAuFoil® holey gold
supports. However, the gold foil is not volatile when glow discharged or plasma
treated, so the grids may be subjected to more extensive plasma treatments thanstandard carbon foils, without any risk of degrading the surface. A good rule of thumbis to approximately double the plasma cleaning/glow discharge times compared toUltrAuFoil® grids. A table of suggested settings is provided below for some common
glow discharge instruments (as originally published in Naydenova and Russo, 2022).

SUGGESTEDPLASMA CLEANING SETTINGS

6PARAMETERFISCHIONE 1070

EDWARDS S150B

TEDPELLAEASIGLOW

ATMOSPHERE

9:1 Ar:O2

Residual air

Residual air

PROCESSPRESSURE

21 mTorr

150 mTorr

0.39 mBar (290 mTorr)

POWER/CURRENT

40 W

30 mA

25 mA

EXPOSURE
TIME

120-180 s

60 s

90 s

SAMPLE PREPARATION PROTOCOL CHANGES CONTINUED

2. VITRIFICATION SETTINGS
In general, HexAuFoil® grids can be directly substituted into your vitrification protocol,
with the exception that blot times should be extended compared to similar samples ona standard larger hole grid. For example, if you currently blot for 10 seconds, with HexAuFoil® grids, you should initially try a 15 second blot. We recommend applying sample to the foil side of the grid and blotting from the same or both sides, but this is sample dependent, and any variation can be used.
Typical settings for vitrification with a HexAuFoil® grid on some common plunge freezing instruments are given below by way of example (taken from Naydenova and Russo, 2022).

SUGGESTEDVITRIFICATION SETTINGSMANUALPLUNGERVITROBOT
MKIV

LEICA
GP2

TEMP

4°C

4°C

4°C

RELATIVE HUMIDITY

100 %

100 %

100 %

WAIT TIME

0s0s0sFORCE SETTING

n/a

10n/a

BLOT TIME

15 s

5s

5s

DRAIN TIME

0s

0s0s

VITRIFICATION MEDIA

EthaneEthaneEthaneVITRIFICATION TEMP

93 K

93 K

93 K

PARAMETER

7HOW DO HEXAUFOIL® SUPPORTS IMPROVEYOUR IMAGES?
1. SMALLER HOLES, THINNER, STABLE ICE
The movement of thousands of gold nanoparticles vitrified on grids with varying holesize was tracked. With smaller hole-sizes, the compressive strain on the ice caused
by local heating from the electron beam was reduced. This in turn led to less bucklingand therefore lower particle movement. With small enough hole size, for a given
ice thickness, buckling is eliminated and particle movement reduces to less than anÅngstrom.
Ce-2.00 o tilt

15

2.2tiora:15ctunstable ratios(buckling)

1.6

pe

hole ø

as

10

rat
io

1.801020
Fluence (e -/Å2)e-20

RMS displacement (Å)

30hole ø

15

pe:1110D ia me te r ( μ m)

as

1.4

ct

15

RMS displacement (Å)

20

1.2
1.0
0.8

stable ratios(no buckling)

0.6

30 o tilt
100.4
DPS

500.2

01020
Fluence (e -/Å2)

30

0.0
0500

1000
Thickness (Å)

1500

2000

By measuring the observed particle movement parallel to the beam, it is possible
to determine a ratio between ice thickness and hole size that determines whether
buckling can occur. Ice buckling at hole diameter:ice thickness ratios of 11:1 or greater.
For biological specimens, where the ice needs to be at maximum 3-400 Å thick, thismeans that hole diameters should be no more than 300 nm, as with HexAuFoil® ultrasmall hole gold sample supports.

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IMPROVING YOUR IMAGES WITH HEXAUFOIL® GRIDS CONTINUED

2. STABLE ICE GIVES HIGHER QUALITY, MORECONSISTENT DATA
The combination of 300 nm holes in a gold film eliminates both buckling and in planebeam-induced motion. Overall, particle motion is reduced to sub-Ångstrom levels thatcan be attributed to Brownian motion-type movement.
HexAuFoil® grids deliver consistent thin ice across large regions of the grid. Thisconsistency combined with increased particle stability delivers reduced re-focussing
and increased consistency in data collection parameters such as defocus and ctf
correction.

20k
10k
00

2k

4k

6k

8k

10k

12k
Index

Defocus (Å)

Defocus (Å)

AVERAGE DEFOCUS (Å)
30k
20k
10k
002k

4k

6k

8k

10k

12k
Index

2k

4k

6k

8k

10k12kIndex

2k4k6k8k

10k12kIndex

4020002k4k6k8k10k12kIndex

Resolution (Å)

Resolution (Å)

CTF FIT RESOLUTION (Å)

402000

RELATIVE ICE THICKNESS

1.4
1.2
102k

4k

6k

8k

10k12kIndex

2.0
1.5
10.5009

IMPROVING YOUR IMAGES WITH HEXAUFOIL® GRIDS CONTINUED

3. EXTRAPOLATE RECONSTRUCTIONS TO ZERO DOSE
With movement at sub-Ångstrom (Brownian motion) levels, B-factors are no longer
dominated by beam-induced motion. Well-developed theory relating B-factor to atomic
motion and radiation damage can therefore be applied, to allow the calculation ofstructure factors and subsequently reconstructions at zero dose. The program 0dose(https://www.mrc-lmb.cam.ac.uk/crusso/resources.html) will shortly be included with
software packages including CCPEM, allows researchers to apply this correction totheir data. Reconstructions at zero dose offer consistency of reconstructions between
datasets, facilitating comparisons between lead compounds in drug discovery as well
as offering improved reconstructions.

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FURTHER INFORMATION ABOUT THE DEVELOPMENTAND USE OF HEXAUFOIL® GRIDSLight-harvesting 2 complex: PDBID 6ZXA, EMDB entry 11516. Structure solved
with data collected using HexAuFoil® grids. As described in Gardiner et al (Sci.
Adv.,2021)

HEXAUFOIL® DEVELOPMENT AND BEST PRACTICE
More details about the development and use of HexAuFoil grids can be found at:

Naydenova and Russo. Integrated wafer-scale manufacture of electron cryomicroscopy specimen supports. Ultramicroscopy 232: 113396 (2022).
Naydenova, Jia, Russo. Cryo-EM with sub-1 Å specimen movement. Science 370: 223-226 (2020).

STRUCTURES SOLVED USING HEXAUFOIL® GRIDS
Naydenova et al. Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence offavipiravir-RTP. Proc. Natl Acad. Sci 118: e2021946118 (2021).
Gardiner et al. The 2.4 Å cryo-EM structure of a heptameric light-harvesting 2 complex reveals two
carotenoid energy transfer pathways. Sci. Adv. 7: e4650 (2021).
Qian et al. 2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem. Sci. Adv. 8:
eabk3139 (2022).

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YOUR INTEREST

Or contact us for more information:
 marketing@quantifoil.com
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