Journal of Structural Biology 215 (2023) 107959

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Journal of Structural Biology

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A review of the approaches used to solve sub-100 kDa membrane proteins

by cryo-electron microscopy
Peter J. Harrison a, b, 1, Tereza Vecerkova a, b, c, 1, Daniel K. Clare b, *, Andrew Quigley a, *
a
Membrane Protein Laboratory, Diamond Light Source, Research Complex at Harwell, Didcot, UK
b
Electron Bio-Imaging Centre, Diamond Light Source, Didcot, UK
c
School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, UK

A R T I C L E I N F O A B S T R A C T

Keywords: Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular
Atomic resolution functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important
cryo-EM biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron
Membrane protein
microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structur­
Protein purification
Structural biology
ally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity
as well as conformational and compositional instability. Here we have reviewed the sample preparation ap­
proaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM
(those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches
towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each
stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss
future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.

1. Introduction structural genomics, sample preparation and X-ray crystallography.

The human proteome comprises of approximately 20,000 unique
proteins, of which around a quarter encode for MPs (Almén et al., 2009;
Uhlén et al., 2015). Not all proteins in the human proteome can be
classified as a suitable pharmaceutical target but of those proteins that
are, the membrane proteome is disproportionally represented with 50%-
60% of all FDA approved small-molecule pharmaceuticals targeting a
human MP (Santos et al., 2017). The first structure of a MP was deter­
mined at 7 Å by electron diffraction and published in 1975 (Henderson
and Unwin, 1975), but it took another 10 years for the first high-
resolution structure to be determined (Deisenhofer et al., 1985). Since
the first MP structures were published there has been an exponential
increase in the number of available structures deposited in the Protein
Data Bank (PDB). As of December 2022, 1534 unique structures have
been deposited according to the ‘Membrane Proteins of Known 3D
Structure’ database. These structures have been solved using either X-
ray crystallography, cryo-EM, electron diffraction or nuclear magnetic
resonance spectroscopy (Fig. 1A). The exponential increase in high
resolution structures of MPs has been driven by developments in
With the beginning of the electron microscopy ‘resolution revolution’ in
2011, there has been systematic shift toward single-particle analysis
(SPA) using cryo-EM for determining the atomic resolution structure of
MPs (Fig. 1B). Fewer than 10% of MP structures were solved using X-ray
crystallography in 2022 compared to 85% of MP structures in 2011. The
cryo-EM resolution revolution has been primarily driven by advances in
microscope, camera and data processing technology, such as the intro­
duction of direct electron detection cameras, in combination with
improved processing software. With the development of computational
algorithms to address the protein folding problem such as AlphaFold
(Jumper et al., 2021), these experimentally derived structures have been
an invaluable tool in the understanding of global structural biology.
Despite the incidence and importance of MPs, they account for only
14% of unique depositions in the PDB (1534 unique structures in the
‘Membrane Proteins of Known 3D Structure’ database compared to
10,712 unique structures in the PDB (December 2022)). MPs are noto­
riously difficult to study as they are intrinsically more unstable (espe­
cially outside of the membrane), express in lower yields and they must
be removed from their native membrane-based environment. Typically,

* Corresponding authors.
E-mail addresses: daniel.clare@diamond.ac.uk (D.K. Clare), andrew.quigley@diamond.ac.uk (A. Quigley).
1
Contributed equally.

https://doi.org/10.1016/j.jsb.2023.107959
Received 20 December 2022; Received in revised form 7 March 2023; Accepted 28 March 2023
Available online 31 March 2023
1047-8477/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
P.J. Harrison et al. Journal of Structural Biology 215 (2023) 107959

this is achieved using detergents, but detergent free systems are
becoming increasingly popular. Cryo-EM can alleviate some of the issues
of MP production, especially the need for large quantities of material,
but challenges remain. For cryo-EM, particle size and conformational
stability are major determinants of success. It is unsurprising therefore,
that the first high-resolution single-particle cryo-EM structures of MPs
were of large multimeric proteins such as TRPV1 (Cao et al., 2013; Liao
et al., 2013). Despite the benefits of single-particle cryo-EM, sub-100
kDa MPs are still challenging targets due to low contrast and signal-to-
noise as well as a lack of features outside the membrane. Of the
13,200 structures solved by cryo-EM only 1159 are under 100 kDa in
size, and of these only 190 are MPs (based on PDB query searches in
December 2022). For the purposes of this review, we have defined a sub-
100 kDa MP as a MP resolved at less than 5 Å resolution, with a total
structural weight that is less than 100 kDa and published in a peer
reviewed journal. A description of the search query parameters can be
found in the Supplementary Information. Sub-100 kDa MPs with a total
solved molecular weight of above 100 kDa (for example by attachment
to a megabody or fab fragment) are not covered by this review, but we
provide a brief discussion of these approaches. Based on these param­
eters we identified 128 unique MP structures in the PDB belonging to 64
target proteins. The data we have collated is available as a Supple­
mentary File.
Sample type, preparation, data acquisition approaches and data
processing choices all require optimisation to determine an atomic res­
olution structure by cryo-EM. Making rational choices at each step in the
process is sometimes opaque and we aim to provide an analysis of the
choices made to solve sub-100 kDa MPs. Our aim is not to provide a
recipe for a successful structural campaign, but to identify trends or
common threads running through these published structures. For ease of
analysis, we have used 1/resolution (Å− 1) to analyse structures. A brief
explanation of our analysis methodology is provided in the Supple­
mentary Information section.
2. Protein family & species origin
There are 77 published unique sub-100 kDa MP studies at a resolu­
tion of below 5 Å. The transporter family contributes the largest fraction
of these unique studies (39), followed by receptors (17), enzymes (11),
channels (6) as well as others (Fig. 1C). In 32 out of a total of 77 studies
of sub-100 kDa MPs structures, apo and ligand-bound states were re­
ported, thus there are 128 structures deposited from 77 published
studies. There is no statistical difference (p > 0.05) in the final resolution
achieved by different classes of proteins (SI Fig. 1A). The bias towards
the numbers of deposited transporter structures may be explained by the
availability of ligands, that may potentially stabilise conformations as

Fig. 1. Analysis of deposited MP structures in the Protein Data Bank. A: Membrane protein PDB depositions (based upon the ‘Membrane Proteins of Known 3D
Structure’ database) since the first atomic resolution MP structure was solved in 1985 by X-ray crystallography. There has been an exponential increase over the last
20 years. In particular, the number of structures solved by cryo-EM since 2011, whilst there is a small contribution from electron diffraction (ED) and Nuclear
Magnetic Resonance spectroscopy. B: Depositions of sub-100 kDa MP structures resolved to less than 5 Å since the first structure was deposited in 2017. A total of 77
unique MPs and a total of 128 conformations have been deposited to date (December 2022) C: Proportion of solved and published sub-100 kDa MP cryo-EM
structures by protein family. D: Proportion of protein species origin represented in sub- 100 kDa MPs solved by cryo-EM.

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P.J. Harrison et al.
well as the ability to select different conformational and oligomeric
states by cryo-EM allowing for higher-level sample heterogeneity
compared to X-ray crystallography.
Structures of sub-100 kDa MPs have been solved from all domains of
life, but the vast majority are of animal origin with human sources ac­
counting for 70 structures, and other mammalian sources a further 22
out of a total of 128 structures. Far fewer sub-100 kDa MPs originate
from bacteria with 18 reported structures. Archaea and viral MPs ac­
count for 6 each and plants and fungi 3 each (Fig. 1D). The shift of focus
to structures of higher order eukaryotic proteins is partly due to
improved methods for protein production in mammalian systems, a
reduction in production costs over the past decade and a focus on human
pharmaceutical targets. There is no statistically significant difference (p
> 0.05) in final resolution achieved between proteins of human, animal
or bacterial origin (SI Fig. 1B).
3. Sample preparation
Without doubt, the provision of usable yields of high-quality protein
is key to any high-resolution structure determination. The careful design
of expression constructs must consider the incorporation of promotor
elements, reporter and purification tags as well as enzyme cleavage sites
that may be used to remove these tags. The target gene sequence may
require codon optimisation, specific mutations, or insertions introduced
to stabilise the MP. The choice of host expression system as well as
purification strategy will have been made prior to construct design un­
less using expression vectors that contain multiple host promoter ele­
ments or vectors with multiple purification tags (3.1 Construct Design,
Expression and Purification). Once expressed, decisions on the use of
detergent (3.2 Detergents) or non-detergent (3.3 Non-detergent encap­
sulation systems) solubilisation and stabilisation approaches are
required and sometimes excess detergent may need to be removed (3.4
Homogenous samples and removing excess detergent). Finally, it may be
necessary to solve the structures as part of a complex (4.0 Binders,
Complexes and Symmetry). These parameters and strategies for sample
optimisation have been well reviewed elsewhere (Birch et al., 2020;
Kesidis et al., 2020).
3.1. Construct design, expression and purification
Our analysis of sub-100 kDa MP structures reveals that most proteins
are expressed in a phylogenetically closely related organism, presum­
ably to ensure close-to-native protein is produced (SI Table 1). Human
expression systems are used for the majority of proteins of human origin
(25 out of 44 human proteins), with the remainder expressed in insect
cell lines (Sf9 and Trichoplusia ni). Seven sub-100 kDa MPs mammalian
proteins were reportedly expressed in human based hosts with a further
four MPs expressed in insect cells and four MPs expressed in fungal/
yeast hosts. There is no effect on the final resolution (p > 0.05) when
expressing human or mammalian proteins in either human or insect cells
lines (SI Fig. 1C). The majority (14 out of 17) prokaryotic proteins are
expressed in Escherichia coli, unless a more closely related expression
system is available, such as Mycobacterium smegmatis (3 out of 17) for
tuberculosis targets. The Saccharomyces cerevisiae expression system has
been used for expression of proteins from fungi (1), plant (1), yeast (2),
and a few mammalian proteins (3).
The poly-histidine-tag (his-tag) is the most popular purification tag
accounting for 36 out of 77 sub-100 kDa MP studies. FLAG and Strep
based tags are the second most popular accounting for 16 and 15 MPs
respectively. Green fluorescent protein (GFP) has been used as a puri­
fication tag in 9 cases, purified with nanobody-conjugated resins. Out of
74 constructs (construct choice not always mentioned in methods), 41
contained more than one purification tag (SI Fig. 1A), but only 7 pro­
tocols utilised more than one tag for affinity chromatography. There is a
small (0.4 Å) statistically significant difference in the final resolution
achieved when comparing the use of a Strep tag (average Å− 1 = 0.26 ±
3
Journal of Structural Biology 215 (2023) 107959
0.03) to a His tag (average Å− 1 = 0.30 ± 0.04) (p = 0.000973). More­
over, there is also a statistically significant difference between the FLAG
tag (average Å− 1 = 0.298 ± 0.04) and the strep tag (p = 0.031). There
was no statistical difference for any other comparison (SI Fig. 3A). This
observation may be due to the fact that Strep-based resin is often used to
purify difficult to obtain or low yielding membrane proteins, so may
indicate that these proteins are intrinsically less stable. The most pop­
ular immobilised metal-ion affinity chromatographic (IMAC) resins
were nickel-based. 27 studies reported using nickel-nitrilotriacetic acid
(Ni-NTA) and a further 3 used unspecified nickel-based resins (SI
Fig. 3B). Five studies used cobalt-based resins. Other resins reported
include Streptactin (14), Flag M1 (9) and M2 resins (7), and anti-GFP
conjugated affinity resins (9). Four other resins, Tse3, an immobilised
DARPin, G1 and Streptavidin resins, were used in single cases (SI
Fig. 3B).
3.2. Detergents
To study MP function and structure, it is often necessary to extract
MPs from membranes into encapsulation agents that mimic lipid envi­
ronment to maintain protein integrity and stability. MPs were tradi­
tionally solubilised using detergents which are still the most popular
choice for cryo-EM studies of sub-100 kDa MPs. Selection of the most
suitable detergent can be time consuming, but there are now numerous
published screening protocols available for optimal detergent selection
(Desuzinges Mandon et al., 2017; Kotov et al., 2019; Vergis et al., 2010).
Given the breadth of detergents available, there is surprisingly little
variation in the detergents used for solubilisation of sub-100 kDa MPs
(Fig. 2A). The popularity of n-Dodecyl-β-D-maltoside (DDM) has per­
sisted from crystallographic studies, due to its low critical micelle con­
centration (CMC), mild nature, and price compared to other detergents.
DDM was used in 47 out of 77 studies demonstrating the robustness of
DDM, its suitability for variety of different proteins and its use as a good
‘first choice’ detergent. However, it may be necessary to screen a range
of detergents before performing cryo-EM analysis. Lauryl Maltose Neo­
pentyl Glycol (LMNG) is another notable detergent with its use reported
in 22 studies.
Detergents are the most popular encapsulation agents for cryo-EM
when purified MPs are applied to a cryo-EM grid (Fig. 2B). The popu­
larity of LMNG is close to that of DDM (11 and 12 out of 44 studies
respectively) among the proteins solved in detergent micelles (Fig. 2C).
This may be due to the low CMC and slow off rate of LMNG, which
means LMNG solubilised proteins are stable even at sub-CMC concen­
trations (Hauer et al., 2015), which can help to reduce the detergent
background in micrographs. Digitonin (5) and its synthetic derivative
glycol-diosgenin (GDN) (7 in GDN and 7 in GDN/LMNG) are also
commonly used during cryo-EM. Given the differences in the biophysical
properties of DDM, DM (n-Decyl-β-Maltoside), LMNG, digitonin and
GDN, it is not possible to say that sub-100 kDa proteins have a prefer­
ence for one over the other (SI Fig. 4A and SI Fig. 4B). There is a clear
supremacy of LMNG and DDM over other detergents and detergent
combinations, which may be biased by the fact they are common ‘first
choice’ detergents.
Protein extraction by detergents from native membrane removes
much of the surrounding lipid environment, which may severely impact
protein integrity. Cholesteryl hemisuccinate (CHS) is commonly used
with detergents (43 out of 77 studies), as it is a more soluble derivative
of cholesterol. Cholesterol is a major component of membranes across
higher eukaryotic species, and so its addition during purification is a
rational step for proteins from species whose membranes natively
contain cholesterol. CHS can help stabilise proteins, and so for many
projects its addition may well be an essential component of the purifi­
cation process to maintain conformational stability (Drew et al., 2008;
Meury et al., 2014). CHS was supplemented during purifications of
mammalian, viral and plant proteins, in fact 74.1% of all mammalian
proteins had CHS added during solubilisation.
P.J. Harrison et al. Journal of Structural Biology 215 (2023) 107959

Fig. 2. Encapsulation agents used for solubilisation and imaging by cryo-EM A: Distribution of detergents used for solubilisation. DDM and LMNG are by far the
most popular solubilisation agents used in 69 out of 77 solubilisation protocols. B: Encapsulation system used when imaging by cryo-EM. Detergents (43 out of 77)
and discs (26) are the most common encapsulation agents for cryo-EM of sub-100 kDa MPs, amphipols (8) are preferentially used for non chaperone-MP complexes
(see section 4.0) and oligomeric structures, and SMALP system was used only in 1 study. C: Detergent used when imaging by cryo-EM. Structures solved in detergent
micelles are most commonly in DDM, LMNG, GDN, or their combinations. D: Disc system used when imaging by cryo-EM. Out of the wide variety of nanodiscs
available, nanodiscs of smaller sized diameters, mainly MSP1D1 and MSP1E3D1 are used for cryo-EM analysis. E: Amphipol used when imaging by cryo-EM.
Amphipols are used mostly for cryo-EM of multimeric proteins, with PMAL-C8 used in 6 out of 8 preparations. F-I: Representation of sub-100 kDa in encapsula­
tion agents: ZnT8 (PDB: 6XPD) in a detergent micelle, GLUT4 (PDB: 6X12) in a nanodisc, Signal peptidase complex paralog A (PDB: 7P2P) in an amphipol, PBP1
(PDB: 7LQ6) in a SMALP system.

3.3. Non-detergent encapsulation systems
Although detergents are excellent at solubilising MPs and preserving
structural features, they can make image processing difficult due to an
empty detergent micelle background and reduced conformational sta­
bility of MPs in detergents over other non-detergent-based encapsula­
tion systems. Nanoscale phospholipid bilayer systems such as nanodiscs
and co-polymer systems provide an alternative to detergents. Some MPs
(GLUT4, PepT1, TMEM120A and the hepatic sodium / bile acid
cotransporter) have been solved both in detergents and in a nanodisc.
There has been a rapid expansion in the reported number of non-
detergent systems available for MP encapsulation. Nanodiscs are the
most popular non-detergent system with 26 studies reporting their use
(Fig. 2D). Other reported non-detergent encapsulation systems include
amphipols, reported in 8 studies, and recently the use of a styrene maleic
acid lipid particle (SMALP) was reported (Caveney et al., 2021).
Nanodiscs are disc-shaped particles composed of lipids and an
amphipathic protein or co-polymer scaffold. Various types and sizes of
nanodisc have been used to study sub-100 kDa MPs allowing a tailored
approach to the individual protein. The first protein based nanodiscs
were synthetically developed from Apolipoprotein A and termed mem­
brane scaffold proteins (MSPs) (Bayburt et al., 2002). Subsequently
other protein-based systems have been developed including peptidiscs
(Carlson et al., 2018) and Salipro (saposin-lipoporotein) (Drulyte et al.,
2023; Popovic et al., 2012). Both the peptidisc and Salipro encapsulation
systems can directly solubilise MPs (Carlson et al., 2018; Lloris-Garcerá
et al., 2020). Styrene-maleic acid lipid particles (SMALPs) are an
example of a co-polymer-based encapsulation system, capable of direct
solubilisation of MPs with native lipids from membranes (Jamshad et al.,
2011; Knowles et al., 2009). SMALP solutions do not need to be sup­
plemented, like detergents, during the purification processes reducing
the costs and potentially the background noise in cryo-EM images
(Pollock et al., 2018). Where Salipro or SMALPs have been used for the
encapsulation of sub-100 kDa MPs, the proteins were reconstituted from
a detergent extraction.
MSP1D1 (~9.7 nm) (Denisov et al., 2004) and MSP1E3D1 (~12 nm)
(Puthenveetil and Vinogradova, 2013) are the most commonly used
nanodiscs for sub-100 kDa MPs with 10 and 8 reported studies respec­
tively, while MSP1E3 (~12.9 nm) (Denisov et al., 2004) has been only
reported four times. Recently two structures were solved using Saposin
A (adaptable size) (Flayhan et al., 2018) as the encapsulation agent (Xu
et al., 2022; Zhang et al., 2022) and one structure has been solved using
the nanodisc NW9 (Niu et al., 2022) (~9 nm) (Nasr et al., 2017). There is
one example of a sub-100 kDa MP (TWIK1) that has been solved both in
MSP1D1 and MSP1E3D1 (Turney et al., 2022). We do not observe that
smaller sub-100 kDa MPs tend towards smaller MSP sizes, and vice
versa. In addition, for sub-100 kDa MPs solved in discs, there is no
correlation between protein size and resolution achieved (SI Fig. 4C).
However, nanodisc selection is often made based on the predicted
number of transmembrane helices, and it is for this reason that we
suspect larger MSP sizes are not represented here (Ritchie et al., 2009).
Lipids are an essential component of the nanodisc system helping to
maintain the MP in an environment more similar to the native mem­
brane. The most common lipid is 1-palmitoyl-2-oleoyl-glycero-3-

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phosphocholine (POPC) (supplemented in 13 out of 26 nanodisc

reconstitution studies) either alone, or more commonly in combination
with other lipids. Even though there is no correlation between nanodisc
type and lipids used, the most common nanodisc lipid composition is a
2:1:1 ratio of DOPE:POPS:POPC (6 reconstitutions). Other less
commonly used lipids were E. coli, brain and liver total lipid extract,
based on the species and expression system to closely represent given
native lipid composition.
Amphipathic polymers (amphipols) (Tribet et al., 1996) are poly­
mers with a hydrophilic backbone that makes contact with the buffer
solution, and hydrophobic side chains which closely interact with
transmembrane domains of MPs. Since their introduction, novel
amphipols with different properties have been developed, such as non-
ionic amphipols (NaPol) (Prata et al., 2000; Sharma et al., 2012), or
zwitterionic PMALs (Nagy et al., 2001). Out of eight MP structures that
have used amphipols, two used the original A8-35 polymer while six
studies report the use of the zwitterionic PMALs. Six of the studies that
used amphipols were of protein complexes, suggesting their potential
benefits for multimeric proteins (Fig. 2E).
The versatility of available encapsulation systems (Fig. 2F-I) allows
accomodation of different protein characteristics and requirements.
There is no significant difference (p > 0.05) in final resolution achieved
for different encapsulation systems (detergent Å− 1 = 0.29 ± 0.04, disc
Å− 1 = 0.29 ± 0.044 and amphipol Å− 1 = 0.26 ± 0.06 (Fig. 3A). Within
detergents there is no variation in the final resolution (Fig. 3B). Within
nanodiscs, there are only sufficient data to compare MSP1D1 and
MSP1E3D1, and there is no statistically significant difference in reso­
lution obtained between them.

3.4. Homogenous samples and removing excess detergent

During the purification process, detergent exchange is often under­

taken as the solubilisation detergent is not always the most appropriate
for cryo-EM analysis due to stability, yield and cost of the original
detergent. Out of 44 studies which solved structures in detergent, the
extraction detergent was exchanged, or at least supplemented, in 24
preparations. Exchanges usually took place during size exclusion chro­
matography (SEC). The most common SEC resin for sub-100 kDa MPs is
Superdex 200 Increase as reported in 34 studies. Superdex 200 was re­
ported 16 times while Superose 6 Increase resin and Superose 6 resin
were reported 14 and 8 time respectively (SI Fig. 5). SEC Columns such
as Sepax SRT-C SEC-300 (1), Biorad650 (1) and ENrich SEC 650 (2) were
used to a lesser extent. Column selection is often a laboratory-based
Fig. 3. Encapsulation agent versus the final resolution achieved by each
equipment selection factor rather than inherent (un)suitability for MP
sub-100 kDa protein structure. A: Comparison of encapsulation agent type
purification; however, selection of SEC media can affect the stability of a with the final resolution achieved. The final resolution achieved is not affected
MP during SEC especially since columns such as SRT-C may offer by the encapsulation system used (p > 0.05, SMALP (1) structure were excluded
reduced detergent and hydrophobic protein adsorption. Hi-Trap ion from the analysis). B: Comparison of detergent type with the final resolution
exchange columns were used only in 2 out of 77 studies as a purification achieved. Detergent selection does not affect the final resolution achieved by
step. Techniques such as gradient centrifugation can also be used to sub-100 kDa proteins solved in detergent micelles (p > 0.05, DDM/LMNG (3)
remove excess detergent before vitrification (Hauer et al., 2015). and digitonin/sodium cholate (1) structures were excluded from the analysis).

4. Binders, complexes & symmetry
Cryo-EM data processing of sub-100 kDa MPs is challenging and
crucially dependent on the quality of the primary data. Identification of
particles and image alignment of sub-100 kDa MPs can be improved
through the use of native protein ligands to form larger complexes.
These MP complex structures are important for understanding protein
function, interactions and cellular pathways and have the additional
benefit of increasing the size and mass of the target which can help with
data processing. Additionally, chaperone-MP complexes can be formed
where the protein of interest is bound to nanobody or Fab fragments.
The addition of nanobodies or Fab fragments has two benefits for
structural determination of small proteins; adding mass to the MP target
(Uchański et al., 2020; Uchański et al., 2021) thus aiding particle
picking and potentially locking proteins in a fixed conformation. This
reduces the intrinsic flexibility and heterogeneity of a sample, making
downstream data processing easier. For MPs, it can often be difficult to
resolve the transmembrane helices as they can be occluded by the
detergent micelle or encapsulation agent. This can be problematic for
sub-100 kDa MPs which do not have any soluble domains. Megabodies
(nanobodies with large soluble domains) are frequently also used as they
give a large increase in the mass of the target. This mass increase is often
to over 100 kDa in size, and so proteins solved with bound megabodies
are beyond the scope of this analysis.
Of the 128 sub-100 kDa MP structures that we identified, 30 were
monomeric structures, 28 were part of a homeric complex and 70 were
part of a heteromeric complex. Structural chaperone-MP complexes,
defined as a MP bound to either a nanobody, sybody (laboratory evolved
nanobody) or Fab fragment consisted of 54 of the 70 heteromeric

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P.J. Harrison et al. Journal of Structural Biology 215 (2023) 107959

structures (Fig. 4A). Breaking down the structural chaperone-MP com­

plex classification further revealed the use of Fab fragments, nanobodies
and sybodies 33, 18 and 3 times respectively. 15 structures out of the 70
heteromeric structures were in complex with either another MP or a
soluble protein that was not a structural chaperone. One further struc­
ture was a combination of a heteromeric MP complex and a structural
chaperone (7TDN, which was solved in complex with a Fab). The
remaining 28 sub-100 kDa homomeric MP structures consisted of 26
homodimeric and 2 homotrimeric structures (Fig. 4A). There is no sta­
tistical difference (p > 0.05) between the final resolution achieved be­
tween monomeric, homodimeric and homotrimeric structures. It is
worth nothing, that two proteins have been solved with and without
nanobodies. PepT2 achieved a Å− 1 of 0.29 (7NQK) in the presence of
nanobody and 0.24 (7PMY) in the absence of a nanobody (both in DDM/
CHS). Similarly, the folate carrier/transporter achieved Å− 1 of 0.30,
0.26 and 0.28 (7TX6, 7TX7 & 8DEP, in digitonin) and 0.31 and 0.3
(7BC6 & 7BC7, in DDM/CHS) without a nanobody. A third protein, the
hepatic sodium/bile acid cotransporter, has been solved with both a Fab
(7VAD, 7VAE, 7VAF, 7VAG & 7WSI, Å− 1 of 0.3, 0.29, 0.28, 0.32, 0.30
and 0.30) and a nanobody (7FCI, Å− 1 of 0.27). There are not enough
data to draw any conclusions as to whether nanobodies or other binders
improve resolution (Fig. 4A).
As previously mentioned, soluble domains can be beneficial for
image alignment. Of the 70 heteromeric complex structures, only 4 did
not have a soluble domain (and thus were fully embedded within the
membrane) (Fig. 4B). Of the 58 monomeric or homomeric structures, 17
did not have a soluble domain. For most structures, a small-molecule or
metal ion binder was reported. There are insufficient data to make a
statistical comparison as to whether this affects the final resolution
achieved. Small molecules may have the same effect as nanobodies by
reducing conformational flexibility. Interestingly, there is no correlation
between the size of the solved structure and the resolution achieved
(Fig. 4C).
From our analysis of sub-100 kDa MPs there is no significant dif­
ference (p > 0.05) in the final resolution achieved for structures solved
in C1 symmetry (100 out of 130 structures, Å− 1 = 0.29 ± 0.04) or C2
symmetry (28 of 130 structures, Å− 1 = 0.28 ± 0.04) symmetry (SI
Fig. 6A) (all but two structures analysed were solved in either of these
two space groups). As expected, structures in C2 symmetry had on
average fewer particles in their final reconstructions than those in C1
(on average 180,816 ± 158,424 particles versus 305,706 ± 254,798
particles) (p = 0.018) (SI Fig. 6B). The two structures in C3 were solved
with even fewer particles (87,865), but there are too few structures to
make an accurate comparison.

5. Grid preparation approaches

For SPA, most grids (~95%) are prepared using a ThermoFisher

Scientific Vitrobot (122 out of 128 solely used a Vitrobot), wherein the
protein sample is applied to a grid, blotted between two sheets of filter
paper, and plunged into liquid ethane. The entire process can take up­
wards of 15 seconds, which increases the chances of proteins denaturing
or adopting a preferred orientation on the grid. A handful of researchers Fig. 4. Soluble domains, chaperone-MP complexes, non chaperone-MP
have used either a Leica GP2 or Gatan Cryoplunge, the former uses complexes and protein size versus reported final resolution. A: Compari­
single-sided blotting and is the preferred option for blotting cellular son of the final resolution achieved for monomeric MP structures, homodimeric
samples. All three instruments blot away excess sample before rapidly MP structures, non chaperone-MP heteromeric complexes and chaperone-MP
plunging into liquid ethane to vitrify the sample in the grid holes. As heteromeric complexes (Fab, nanobody or sybody). B: Comparison of the
such, it is difficult to critique new developments in plunge freezing de­ final resolution achieved for heteromeric complexes (chaperone-MP and non
vices (Weissenberger et al., 2021). chaperone-MP), monomeric and homomeric (homodimeric and homotrimeric)
MPs with and without soluble domains. Most MPs solved either have a soluble
Grid choice is an important factor to consider. EM grids are generally
domain or a protein ligand which provides a soluble domain. There is no sta­
made of either copper or gold supports, with a film (usually amorphous
tistically significant difference between the final resolution achieved. C: Rela­
carbon) on top (Thompson et al., 2016). The film contains holes in which tionship between protein size and the achieved final resolution of the structure.
the protein sits. For SPA, small hole sizes are considered to be preferable, There is no correlation between the two.
as this reduces ice buckling and sample movement (Naydenova et al.,
2020). Moreover, smaller hole sizes may prevent over blotting of thin ice
films in the middle of the hole which can lead to clustering at the hole

6
P.J. Harrison et al.
edge, preferred orientation and sample denaturation (Kampjut et al.,
2021). 88% of reported structures are solved using 1.2/1.3 hole grids
with the remaining studies using 2/1, 2/2 and 0.6/1 holes (Fig. 5A). Due
to the dominance of 1.2/1.3 grids it is not possible to draw any con­
clusions in comparison to other hole sizes beyond the popularity of 1.2/
1.3 grids.
Traditionally grid supports are made of copper due to its high ther­
mal conductivity. Gold grids were introduced primarily for cellular to­
mography as gold is non-toxic to cells. Recently, UltrAUfoil grids where
the holey carbon film has been replaced by a thin holey gold film were
developed (Russo and Passmore, 2014; Russo and Passmore, 2016). Our
analysis, with the caveat that fewer studies report the use of UltrAUfoil
grids, indicates that there is no significant difference in final resolution
achieved when comparing carbon on copper grids with carbon on gold
or UltrAUfoil, or when comparing UltrAUfoil grids with carbon on
copper grids (Fig. 5B). A comparison of UltrAUfoils against carbon on
gold grids indicates a statistically significant difference (p = 0.028) in
the final resolution achieved. There are however two statistical outliers
7RXH (0.43 Å− 1) & 7UL2 (0.42 Å− 1) and we note that these two data­
points achieved the highest resolution of any structures in our sub-100
kDa membrane protein dataset. These high resolution datapoints show
the potential gains to be had from improvements to EM technology and
processing. The gold film, coupled with the gold grid support, reduces
Fig. 5. Grid choice for sub-100 kDa MPs. A: Distribution of grid hole sizes
used for sub-100 kDa MPs. A hole size of 1.2/1.3 is by far the most common. B:
Distribution of the final resolution achieved using different types of grid foils.
Carbon on Gold grids are the most popular choice. There is a statistically sig­
nificant difference in final resolution achieved when comparing UltrAUfoil
grids with carbon on gold grids. There is no significant difference in the final
resolution achieved for any other comparison of grid supports and foils.
7
Journal of Structural Biology 215 (2023) 107959
beam induced sample motion and improves the distribution of the
particles on the grid (Rice et al., 2018). These factors should aid struc­
tural determination.
Sample concentration is an important consideration when making
grids as it is necessary to ensure that there are enough particles in the
holes, whilst not overcrowding the grid. Ultimately the best concen­
tration to use is sample dependent. Where a value is given (for a range,
average value was taken), most proteins are vitrified within a range of 4
to 7 mg mL− 1 (51 out of 115), followed closely by 1 to 4 mg mL− 1 (41 out
of 115). Small proteins with lower molecular weight will have more
molecules than of a larger protein at a given concentration (in mg mL− 1),
so it is unsurprising that nine structures report a concentration below 1
mg mL− 1, although seven structures resulted from MPs frozen at con­
centrations above 10 mg mL− 1 (SI Fig. 7). GPCRs are often frozen at high
concentrations which gives densely packed particles in the foil holes
(Danev et al., 2021), saturating the carbon film and forcing particles into
the holes.
6. Data collection approaches
When collecting single-particle data for high-resolution analysis of
any protein, there are several considerations to be made including the
microscope acceleration voltage, the detector and the dose. For sub-100
kDa MPs it is essential to maximise the signal-to-noise ratio. Our analysis
considers acceleration voltage, detector choice, pixel size, collection
settings (super-resolution, Correlated Double Sampling (CDS)), energy
filter slit width, total dose and number of fractions. SPA data collection
is performed using different software packages such as EPU (Thermo­
Fisher), SerialEM (Cheng et al., 2016; Mastronarde, 2005; Tan et al.,
2015), Leginon (Suloway et al., 2005), AutoEMation (Lei and Frank,
2005) or Latitude (Gatan). SerialEM is the most used (56%), automated,
SPA data collection software for sub-100 kDa MPs (SI Fig. 8).
86% of near-atomic resolution, sub-100 kDa, MP cryo-EM structures
were solved using data collected on a 300 keV microscope. 300 keV
microscopes are widely considered the best approach for obtaining high-
resolution data due to greater sample penetration, decreased inelastic
scattering and decreased sample charging relative to 200 keV in­
struments (Wu et al., 2020). However, at lower acceleration voltages,
the elastic cross-section is greater (2.01-fold greater at 100 kV vs. 300
keV) (Peet et al., 2019). This increases the contrast, at the expense of
radiation damage which only increases 1.57-fold at 100 keV compared
to 300 keV. As such, imaging smaller objects at lower acceleration
voltages should be preferable but is not routine as detectors are opti­
mised to work at 300 keV.
Pixel size dictates the maximum final resolution that is possible in a
reconstruction. For ease of analysis we have grouped pixel sizes into 5
groups (~0.5 Å/px, ~0.65 Å/px, ~0.83 Å/px, ~1.1 Å/px and ~ 1.3 Å/
px). High-resolution data collections typically use a pixel size of ~ 1 Å/
px (Feathers et al., 2021). In our analysis of 128 sub-100 kDa MP
structures, a pixel size of ~ 0.83 Å/px was most frequently reported (61
of 128 structures) (SI Fig. 9). Pixel sizes of ~ 1.1 Å/px were the next
most common (46 of 128 structures). The highest resolution recorded for
a sub-100 kDa MP in our dataset is 2.3 Å (7RXH) (at a pixel size of 1.06
Å/px) (Falzone et al., 2022) and from the structures we analysed, an
average final resolution of 3.52 ± 0.50 Å was obtained. There is a pos­
itive correlation between the fraction of Nyquist achieved (Pearson’s r
= 0.78) for a structure and the pixel size data was collected at (data
collected at a larger pixel size is solved closer to the Nyquist frequency)
(SI Fig. 10A). However, data collected at smaller pixel sizes does not
achieve a higher final resolution than those collected at larger pixel sizes
(SI Fig. 10B) (Pearson’s r = 0.024). This is an interesting observation, as
one might expect the improvement in the detective quantum efficiency
(DQE) that imaging at higher magnifications affords to be beneficial. As
such, it can be argued that there is less to gain from collecting at smaller
pixel sizes and that a pixel size of ~ 1.35 Å/px could be used to maximise
the throughput of data collection.
P.J. Harrison et al. Journal of Structural Biology 215 (2023) 107959

Most structures in our analysis were solved using data collected with
either a Gatan K2 (28 of 128) or K3 (93 of 128) direct detection camera.
The K3 camera has an improved frame frate in comparison to the K2,
which reduces coincidence loss and improves detector performance. Our
analysis shows that structures solved with a K3 camera achieve a better
final resolution than those solved with a K2 camera (p = 0.001) (SI
Fig. 11). This is unexpected since there is no significant difference in the
DQE between the K2 and K3 cameras. It should be noted that the K2 is an
older camera, so structures solved with a K3 benefit from improvements
in data processing. The biggest advantage of the K3 is data collection
throughput. A K3 detector is capable of in excess of 1000 movies per
hour (Sheng et al., 2022). There are not enough data to make a com­
parison between Falcon 3 and Falcon 4 cameras. Interestingly, our
analysis suggests that more data is not necessarily linked to an
improvement in resolution. The average number of final particles is
276,801 (with a standard deviation of ± 241,346 which reflects the
variation in the number of final particles used in reconstructions) from
an average of 10,124 ± 8081 movies. We see a slight positive correlation
between the number of particles in the final reconstruction (Pearson’s r
= 0.31) (Fig. 6A). There is no significant correlation between the initial
number of particles picked (Pearson’s r = 0.22) or the number of movies
collected (Pearson’s r = -0.11) and the resolution achieved in the final
reconstruction (Fig. 6B and Fig. 6C). We see a weak positive correlation
(Pearson’s r = 0.24) between the number of movies collected and the
number of final particles in the reconstruction (SI Fig. 12), suggesting
that more movies does not result in higher resolution structures, and
further emphasises the need for high quality sample preparation.
Energy filtered transmission electron microscopy (EFTEM) removes
inelastically scattered electrons, which improves contrast and signal to
noise (Langmore and Smith, 1992; Schröder et al., 1990; Yonekura et al.,
2006; Zhu et al., 1997). This should be of significant benefit to sub-100
kDa MPs. It should be noted that the greatest effect of energy filtering is
for thicker samples (Yonekura et al., 2006), and thus for small objects in
thin ice the amplitude contrast enhancement will be less pronounced.
Where a slit width was reported (62 cases), 73% used a slit width of 20
eV (SI Fig. 13). Slit widths of 10 eV, 15 eV and 25 eV were also reported.
The current width of energy filter slits used is dictated by their stability
as drift of the zero-loss peak can result in a loss of electron transmission
to the detector. As new energy filters become available that have greater
stability, smaller slit widths will become more accessible, thus the signal
to noise achievable should improve further.
Many reported structures have been solved using ‘super-resolution’
mode. Super-resolution is a detector feature which allows a single
electron event to be sub-localised within a quadrant of a single pixel
(Booth, 2012; Li et al., 2013). Reconstructions may subsequently go
beyond the physical Nyquist limit dictated by the pixel size. From our
analysis, there is no significant difference in final resolution achieved for
structures in normal resolution versus in super-resolution, indicating
that there is less of a benefit for sub-100 kDa MPs (SI Fig. 14). A small
handful of structures were collected in CDS mode (Sun et al., 2021). CDS
mode, samples each pixel’s voltage twice per frame (at the start and at
the end), with the initial value subtracted from the final value, which
increases ‘the detectability’ of an electron, improving signal to noise
which in turn should result in higher-resolution reconstructions, espe­
cially for small proteins which are often difficult to see. Unfortunately,
in our dataset there were only three collections done in CDS, therefore
we cannot make any inferences on the effectiveness of CDS for sub-100
kDa MPs. It will be interesting to see if this feature becomes more readily
Fig. 6. Initial and final number of particles and number of movies versus
used. One disadvantage of CDS is the reduction in relative throughput,
final resolution A: Relationship between the final number of particles in each
as due to the two read per frame (750 fps versus 1500), lower electron
reconstruction, B: the initial number of particles picked and C: the number of
counts must be used to avoid coincidence loss. movies collected versus the final resolution achieved for each structure. There is
Total dose applied to a sample for imaging is an important consid­ a low correlation between either of the variables and the final resolu­
eration. The electron dose experienced by a sample is a function of the tion achieved.
sample itself as well as the energy, fluence and rate at which electrons
are delivered. In the field of electron microscopy electron dose and
electron fluence are often used interchangeably (Ilett et al., 2020) and

8
P.J. Harrison et al.
thus dose is usually recorded as the number of electrons per Å2 (e/ Å2).
This is effectively a measure of the ‘dose over vacuum’. A higher total
dose should give better signal at lower spatial frequencies, increases
contrast and gives a better signal-to-noise in the micrographs, thus
making it easier to see small objects. Higher electron doses come at the
expense of beam induced sample damage with high-resolution sample
information lost first (Cheng et al., 2015; Grant and Grigorieff, 2015).
Beam induced damage is alleviated through dose-weighting of the mo­
tion corrected micrographs (Grant and Grigorieff, 2015; Zheng et al.,
2017). The average dose applied is 56.3 ± 10.4 e/Å2 (SI Fig. 15A) and
there is no significant correlation between applied dose and the final
resolution achieved (Pearson’s r = 0.18) (SI Fig. 15B).
As discussed, a key issue with imaging small proteins in the TEM is
that of contrast. Smaller samples have less scattering material, reducing
contrast, and making the identification of particles much harder. In
microscopy, phase plates are used to introduce additional phase shift to
the scattered or un-scattered electrons depending on the type of phase
plate (or photons in the case of light microscopy). This additional phase
shift increases the phase contrast in the image making it easier to
identify and subsequently align your protein of interest (Wang and Fan,
2019). In biological cryo-EM, the most commonly used phase plate is the
Volta phase plate (VPP) (Danev et al., 2017; Danev et al., 2014). Despite
its promise, the VPP is not routinely used for SPA in Cryo-EM. Of the
structures we examined, only three studies (6 structures in total) used a
VPP (Shang et al., 2019; Xue et al., 2020; Zhao et al., 2019). The highest
resolution of these structures was reported at 3.8 Å. Practically, the VPP
is difficult to align and operate, has limited reproducibility and makes
fitting and correction of CTF harder (Wang and Fan, 2019). These dif­
ficulties, coupled with the fact the phase plate film absorbs approxi­
mately 10% of electrons thus leading to signal loss, help explain their
limited use. This may change in the coming years when the next gen­
eration of laser phase plates come online.
7. Data processing approaches
A final part of the pipeline for structural determination by cryo-EM is
data processing. Software choice for motion correction and CTF esti­
mation is a personal choice. From our analysis, motion correction was
most commonly performed in MotionCorr2 (99 out of 128 structures)
(Zheng et al., 2017). However, other motion correction software such as
Unblur (Grant and Grigorieff, 2015), and implementations in Relion and
CryoSPARC are also commonly used (Rubinstein and Brubaker, 2015;
Zivanov et al., 2018). Similarly, CTF correction most commonly uses
either GCTF (39 out of 128 structures) (Zhang, 2016) or CTFFIND (60
out of 128 structures) (Rohou and Grigorieff, 2015) or an implantation
in Relion or CryoSPARC (Zivanov et al., 2020).
Following motion correction and CTF estimation, particles must be
picked from the micrographs. Generally picking software can be divided
into 3 discrete categories: Template pickers, neural networks (where a
small subset of picked particles is used to train a model such as crYOLO
(Wagner et al., 2019) or Topaz (Bepler et al., 2020)) and Gaussian
pickers such as DoG picker (Voss et al., 2009). Often with small objects it
is difficult to distinguish particles on a micrograph before class aver­
aging, so it is essential to ensure particle picking is as accurate as
possible and that particles, in several different orientations, are selected
from the micrographs. Typically, a small set of several thousand parti­
cles are picked either manually or using software such as Blob Picker.
Selected particles are then used to train a model for automated picking.
Alternatively, 2D classes generated from the initial picking can be used
as templates for the autopicking software. From our analysis we have
observed that there are multiple software options and a high variability
in approaches taken. It is not possible to quantify which software
package or approach is the most successful.
2D classification, initial model generation, 3D classification, 3D
refinement and polishing are generally performed using either Relion or
CryoSPARC (Punjani et al., 2017; Zivanov et al., 2018). In fact, all of the
9
Journal of Structural Biology 215 (2023) 107959
structures we analysed at some point used either Relion or CryoSPARC.
In 41 of the 128 structures analysis here, a mixture of both software
platforms were used. Bayesian polishing (Scheres, 2012) of the final map
is often performed in Relion. From our analysis of solved sub-100 kDa
MP structures we do not find that there are definitive trends towards a
particular software type or feature. It is worth noting that the majority of
the processing pipelines use either CryoSPARC or Relion, but there may
still be some advantages to trying other software such as Sphire (Au -
Moriya et al., 2017) or cisTEM (Grant et al., 2018) for cases where other
software struggles.
8. Further developments
At the time of writing this review there are a handful of MP structures
in the region of ~ 44 kDa (excluding the size of the encapsulation agent)
deposited in the PDB, demonstrating that the limits of single particle
cryo-EM are being continually pushed. Future developments in micro­
scope, detector and image processing software are likely to decrease the
lower limit of solvable structures both in SPA and cryoET even further.
Future developments in microscope architecture have been nicely
summarised in a recent review by Russo et al. (Russo et al., 2022). Im­
aging at liquid helium temperatures potentially offers benefits in
decreased radiation damage and improved signal to noise (as a higher
electron dose can be applied to the sample) (Naydenova et al., 2022;
Pfeil-Gardiner et al., 2019; Russo et al., 2022). However, due to
increased sample movement in liquid helium cooled samples, these
gains are currently out of reach (although improvements in image pro­
cessing software are likely to help offset this). Chromatic aberration
correctors which can focus inelastically scattered electrons and laser
phase plates could both help to increase signal and contrast in cryo-EM
images (Dickerson et al., 2022; Schwartz et al., 2019). As discussed,
imaging at 100 keV could also have benefits for small MPs, if suitable
detectors become available. It remains to be seen whether these im­
provements in microscope technology lead us to a concurrent increase in
resolution.
‘Artificial intelligence’ or deep learning techniques are already being
applied to the image processing pipeline (Chung et al., 2022). Currently
they are mostly used for particle picking, with deep learning software
such as crYOLO, Topaz and Warp (Tegunov and Cramer, 2019) already
commonly used. Tools such as CryoDRGN (Zhong et al., 2021) and
3DFlex (Punjani and Fleet, 2022) are also available to aid with hetero­
geneity in the 3D reconstruction phase and DeepEMhancer (Sanchez-
Garcia et al., 2021) are aiding with map sharpening. Given the
increasing complexity of cryo-SPA data sets due to intrinsic protein
flexibility and complexes rather than individual proteins becoming
targets, deep learning tools for model building, classification and
refinement will become crucial.
For SPA cryo-EM of sub-100 kDa MPs, new polymer reagents such as
SMALPs or Salipro may aid in the solubilisation stage of sample prepa­
ration with maintaining near-native lipid environment. With the
development of large nanodiscs and peptidiscs (allowing adaptability to
protein size) (Angiulli et al., 2020) the ability to capture larger com­
plexes will open up the possibility to image large membrane complexes.
As technology improves, we will move to a situation where more MPs of
smaller size can be imaged in cellulo by cryo-electron tomography,
(species smaller than 500 kDa are considered small for CryoET) (Turk
and Baumeister, 2020). Similarly, developments in labelling and func­
tionalisation of proteins will be crucial for this. Moreover, development
of time-resolved EM techniques will allow structural dynamics to be
viewed at atomic resolution (Kelly et al., 2022).
9. Conclusions
Our analysis has provided some interesting insights. For example,
there is no correlation between sub-100 kDa MP size and resolution
achieved, but there is a weak positive correlation between the final
P.J. Harrison et al.
number of particles and the resolution achieved. Most sub-100 kDa MPs
solved by cryo-EM are encapsulated in detergent with DDM the most
popular. Carbon on gold grids are by far the most popular for sub- 100
kDa MPs while 1.2/1.3 grids are almost exclusively used. Although the
basics of data processing are fundamentally the same, many different
approaches are taken for particle picking, 2D and 3D classification as
well as refinement.
For our analysis we have the final resolution reported for each
structure (Å− 1). We have also performed a parallel analysis (see Sup­
plementary Information) using ‘fraction of Nyquist’ (FoN), where the
final resolution is divided by the Nyquist frequency of the imaging
conditions. The purpose of this was to try to observe how close structures
were to achieving their theoretical highest resolution based on the pixel
size used. However, there are issues with this metric as one would expect
that collections at smaller pixel sizes will achieve better resolutions by
virtue of the imaging conditions. Interestingly, both metrics produces
identical conclusions in 12 out of 17 cases (see SI). Both of these metrics
are dependent on consistent resolution estimation from processing
software. A better metric may well be the B-factor, as described by
Henderson and Rosenthal (Rosenthal and Henderson, 2003). The B-
factor can be used to estimate the number of particles needed to produce
a reconstruction at any resolution and thus provides estimates the
experimental error for a specific experiment. As such, the B-factor takes
into account multiple factors including sample preparation, data
collection parameters and image processing. Thus the B-factor provides
a better description of the overall experiment (Nakane et al., 2020).
However, B-factors are not always reported, which currently makes it
difficult to use this as a metric and we therefore strongly encourage the
reporting Bfactors with data collection metrics.
Fundamentally, data collection and image processing are limited
unless a protein of high-quality is supplied. Given the variety of different
purification methods, encapsulation agents and binding partners
(nanobodies, antibodies) used, there is not a single encapsulation agent
that works for every protein, or system achieving the best possible res­
olution. As such, it is important to expend time and effort on sample
optimisation before collecting data.
CRediT authorship contribution statement
Peter J. Harrison: Conceptualization, Data curation, Formal anal­
ysis, Writing – original draft. Tereza Vecerkova: Formal analysis,
Writing – original draft. Daniel K. Clare: Conceptualization, Funding
acquisition, Supervision, Writing – original draft. Andrew Quigley:
Conceptualization, Funding acquisition, Supervision, Writing – original
draft.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Andrew Quigley reports financial support was provided by Wellcome
Trust.
Data availability
The data used in this paper is freely available in the Protein Data
Bank. A curated version of the results from our search query is made
available in the supplementary materials.
Acknowledgements
We would like to thank colleagues in eBIC, MPL and MX at Diamond
Light Source for helpful discussions and comments. We also thank
Daniel Hatton (Diamond Light Source) for helpful discussions. The
Membrane Protein Laboratory is funded by grants from the Wellcome
Trust (202892/Z/16/Z and 223727/Z/21/Z) with additional support
10
Journal of Structural Biology 215 (2023) 107959
provided by Diamond Light Source and the Research Complex at Har­
well. We also acknowledge the Diamond Light Source for access and the
support of the cryo-EM facilities at the UK national electron Bio-Imaging
Centre, funded by the Wellcome Trust, MRC and BBSRC. Graphical ab­
stract and Fig. 2 were created with BioRender.com.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jsb.2023.107959.
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