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Membrane protein structures without crystals, by singleparticle electron cryomicroscopyKutti R Vinothkumar

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It is an exciting period in membrane protein structural biology

with a number of medically important protein structures

determined at a rapid pace. However, two major hurdles still

remain in the structural biology of membrane proteins. One is

the inability to obtain large amounts of protein for crystallization

and the other is the failure to get well-diffracting crystals. With

single particle electron cryomicroscopy, both these problems

can be overcome and high-resolution structures of membrane

proteins and other labile protein complexes can be obtained

with very little protein and without the need for crystals. In this

review, I highlight recent advances in electron microscopy,

detectors and software, which have allowed determination of

medium to high-resolution structures of membrane proteins

and complexes that have been difficult to study by other

structural biological techniques.

Address

Medical Research Council Laboratory of Molecular Biology, Francis

Crick Avenue, Cambridge CB2 0QH, United Kingdom

Corresponding author: Vinothkumar, Kutti R (vkumar@mrc-

lmb.cam.ac.uk)

Current Opinion in Structural Biology 2015, 33:103–114

This review comes from a themed issue on Membranes

Edited by Bruno Miroux and Eva Pebay Peyroula

For a complete overview see the Issue and the Editorial

Available online 01st October 2015

http://dx.doi.org/10.1016/j.sbi.2015.07.009

0959-440/# 2015 The Author. Published by Elsevier Ltd. This is an

open access article under the CC BY license (http://creativecom-

mons.org/licenses/by/4.0/).

IntroductionMembranes and membrane proteins have always fascinat-

ed electron microscopists because they were prominent

features in the early electron micrographs of the cells. In

the early days, electron micrographs were used to infer the

organization of the lipid and protein components and for

postulation of the fluid-mosaic model of membrane struc-

ture [1]. Specialized membranes with abundant proteins

were also identified by electron microscopy, often with the

constituent proteins arranged in crystalline arrays [2,3].

This paved way for a separate discipline within electron

microscopy, which was later called electron crystallogra-

phy. The pioneering work of Henderson and Unwin on

glucose embedded two-dimensional (2D) crystals of bacte-

riorhodopsin (also called purple membrane) revealed the

first architecture of an integral membrane protein [4,5].

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Subsequent development of the rapid freezing of speci-

mens in thin aqueous films gave rise to the field of electron

cryomicroscopy (cryoEM) [6��,7]. In combination with the

introduction of field emission guns and better vacuums in

the electron microscopes, this led to a great expansion of

cryoEM, including an atomic model of bacteriorhodopsin

[8,9]. It was realized at the same time that membrane

proteins in presence of lipids can also form helical tubes,

which by helical reconstruction can also provide high-

resolution maps as shown for the nicotinic acetylcholine

receptor (AChR) [10]. Thus, in the 90s when obtaining

well-diffracting three-dimensional (3D) crystals of mem-

brane proteins for X-ray crystallography was still difficult,

electron microscopy of membrane proteins in the form of

2D crystals or helical tubes was seen as an alternative

approach to obtain structures of membrane proteins.

The added advantages of the native environment and

the ability to study conformational changes as shown for

bacteriorhodopsin and AChR [11,12] prompted many

researchers to pursue electron crystallography. However,

it soon became clear that obtaining well-ordered 2D crys-

tals or tubes were just as difficult as obtaining well-ordered

3D crystals. Although many membrane proteins formed

2D crystals immediately, often they were poorly ordered

resulting in medium to low-resolution maps, with only a

handful of structures determined to high-resolution [13].

With the advent of genome sequence determination and

identification of homologues of many important membrane

proteins, there has been a rapid increase in structures of

membrane proteins mainly by 3D crystallization and X-ray

crystallography, superseding electron crystallography [14].

Obtaining sufficient membrane protein and well-diffract-

ing crystals still remains an obstacle, and having worked

on membrane proteins for a while, I have always won-

dered if it is possible to completely get away from crystals.

Two techniques that have the potential to provide high-

resolution structures of membrane proteins (or any mac-

romolecule) without crystals include nuclear magnetic

resonance spectroscopy (NMR) and single particle

cryoEM. Of these, structural determination of membrane

proteins by NMR requires a large amount of labeled

protein and stability in a given detergent for at least

few days during measurement [15]. For many membrane

proteins, in particular those from eukaryotes this may not

be feasible. In single particle cryoEM, membrane pro-

teins in solution are rapidly frozen and imaged with

electrons as single molecules in a thin film of buffer.

By averaging a large number of particles, high-resolution

structures can then be obtained with very little protein.


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104 Membranes

This technique has an added advantage that distinct

structural states in a solution can be computationally

separated and multiple structures of the same protein

determined.

Single particle EMElectron micrographs of single protein molecules contain

projections of the underlying structure of the specimen and

due to the limited electron dose required to minimize

radiation damage have a poor signal to noise ratio. Averag-

ing many projections increases the signal and from these it

is possible to generate a three-dimensional 3D reconstruc-

tion (see Figure 1 for examples of electron micrographs and

Figure 1

(a)

Complex I (cymal-7)

C

γ-secretase

γ

R

(b)

Membrane protein imaging as single particles by electron cryomicroscopy.

detergent (Complex I), amphipol (g-secretase) and nanodisc (ryanodine rece

Falcon II detector and the g-secretase was imaged with the Gatan K2 Sum

fluorinated octyl maltoside was added to the protein solution just before fre

seen in these images is due to the use of relatively high dose and sufficient

(72 frames) at a total dose of �68 electrons/A2. This high dose image was o

For subsequent refinements the last 40 frames were discarded [58�]. (b) Re

ryanodine receptor. The panel shows a selection of 2D class averages of di

around the protein (visible as a band around the membrane part and marke

domain (marked with red arrow) and the quaternary structure. In the case o

be seen in the class averages. The box sizes in the 2D class averages are 2

pixel) and ryanodine receptor (1.45 A/pixel) respectively.


class averages). The basic computational principle in single

particle cryoEM involves accurate determination of five

parameters: namely the three Euler angles and the x,ytranslations. A simple rule suggests that the more accurate

the orientation of the particles, the higher the resolution.

Often, this depends on various parameters including the

signal to noise ratio, orientation distribution of the particles

that is if all possible views of the molecule are observed,

and structural heterogeneity. One or all these parameters

affect the achievable resolution. From theoretical estimate,

Henderson has calculated that if perfect images of macro-

molecules can be obtained from an electron microscope

then high-resolution structures can be reconstructed for a

omplex I

(amphipol)

-secretase

Ryanodine receptor (nanodiscs)

yanodine receptor

Current Opinion in Structural Biology

(a) Selected areas of micrographs of membrane proteins observed in

ptor). The images of Complex I and RyR were taken with the FEI

mit detector. Scale bar is 400 nm. In the case of RyR receptor,

ezing to get better distribution of the receptor [38�]. The high contrast

defocus. For example, the Complex I images were captured for 4 s

nly used for particle picking and contrast transfer function estimation.

ference-free 2D class averages of Complex I, g-secretase and

fferent membrane proteins revealing the prominent detergent/lipid belt

d with white arrow), transmembrane (TM) helices in the membrane

f Complex I, the location of some of the supernumerary subunits can

80, 90 and 384 pixels for Complex I (1.72 A/pixel), g-secretase (2.8 A/


Membrane protein structures without crystals Vinothkumar 105

wide range of macromolecules by averaging only few

thousands of asymmetric units [16��,17�]. He pointed

out that radiation damage, beam-induced movement and

signal to noise ratio in images were the major limiting

experimental factors, which when solved would allow to

obtain perfect images and higher resolutions as predicted

from theory.

The advent of microscopes with coherent beams, better

vacuums and optics allowed the first atomic resolution

maps to be obtained by single particle reconstruction of

highly symmetrical macromolecules with images taken on

film or a charged-coupled device (CCD) [reviewed in 18].

These maps showed that it was indeed possible to obtain

high-resolution structures without crystals. In the last

couple of years, with the introduction of direct electron

detectors, it is now possible to pursue structural studies of

less symmetrical macromolecules that were otherwise

difficult to determine. The advantages of the direct

electron detectors can be summarized in the following

points: 1) higher detective quantum efficiency (DQE) or

simply higher signal to noise ratio [19]; 2) the possibility

to look and evaluate the quality of the specimen imme-

diately (unlike films, which had to be developed and

checked by optical diffraction) and collect more data in a

given period of time; 3) as they operate in rolling shutter

mode, the ability to dose-fractionate the exposure allows

one to computationally correct for drift and beam-induced

motion and choose selected frames and electron dose that

gives the best reconstructions [20–24]. There are several

reviews that chart the development of cryoEM from its

early history to the present day advances [25–30,31��],which I highly recommend to readers but this review will

mainly focus on single particle studies of membrane

proteins.

In the early days, electron microscopy of membrane

proteins as single particles was largely limited to visual-

izing them with heavy metal staining. This was based on

the general thought that due to the presence of detergents

it would be difficult to get sufficient contrast in ice.

However, studies on the mitochondrial Complex I [32],

hexameric P-type H+-ATPase from Neurospora crassa [33]

and the bovine F1Fo ATPase [34] showed that single

particle imaging of membrane proteins in ice was possi-

ble. Despite their limited resolution, the calculated maps

were informative and reflected the true structure of

protein (not the envelope from the heavy metal stain).

The structure of bovine ATPase in particular remains a

major landmark as it provided an envelope for an intact

multi-subunit membrane protein complex on which crys-

tal structures of several known subunits could be docked

[35].

Seeing is believing — biochemistry is vitalA great advantage of EM is the ability to directly visualize

the protein of interest and tune the biochemistry to


optimize the specimen for any given investigation. The

homogeneity of membrane proteins is generally judged

based on gels and gel-filtration profiles, occasionally com-

plemented with functional assays. However even a single

band on a gel or a symmetrical gel filtration profile may

not reflect the true nature of membrane protein in solu-

tion. When observing such solutions by cryoEM, one may

sometimes be surprised to see a wide variety of features

including lipid-detergent mixtures either as vesicles or

small membranes, denatured protein or dissociated sub-

units. If the membrane protein of interest is spherical

then it can be difficult to judge the quality of biochemis-

try amid such background. With multi-subunit membrane

protein complexes, the dissociation of subunits can some-

times occur during freezing of protein solution on grids

and some understanding behind the process of blotting

and freezing is thus beneficial [6��]. In the initial stages of

studying a membrane protein by single particle cryoEM,

it is often useful to image them with a combination of high

dose (when using direct detectors) and high defocus, so

that they are clearly visible and some features of the

protein can be recognized. This is particularly useful for

smaller membrane proteins. Such high-contrast low noise

images provide a wealth of information about the purity

and integrity of the sample but also prevent picking

features that could be just low-contrast random noise [36].

Just as in crystallization, the choice of detergent or its

substitutes and the final detergent concentration plays a

critical role in cryoEM. Too high a concentration of

detergent adds to the background. When possible it is

ideal to use the protein directly off the column without a

concentration step. Membrane proteins have been ob-

served as single particles in detergent micelles, amphipol,

liposomes and nanodisc (Table 1 and Figure 1a). To date,

except for liposomes, all other media have produced

medium to high-resolution structures so at present it is

not possible to generalize about whether a particular

detergent or its substitute is best suited for cryoEM or

is essential to obtain high-resolution. The recent struc-

tures of ryanodine receptor (RyR) in detergent [37�],nanodiscs [38�] or mixed lipid-detergent micelles [39�]reveal similar structures and highlight the fact that the

specimen preparation is likely to play a more crucial role

(e.g. thickness of ice) rather than the choice of detergent

or its substitute. For a given membrane protein, it is

important to find the right mixture of detergent/lipid that

keeps in its functional and native state rather than fol-

lowing a particular protocol that has been found to be

successful for other proteins.

Reference free 2D class averaging, where particles with

similar views are averaged to produce projections of the

structure that are less noisy and can further provide assess-

ment of the quality of the protein prep. This requires only a

few thousand particles and perhaps 50–100 images. In

Figure 1b, examples of 2D class averages of different


106 Membranes

Table 1

Compilation of membrane protein structures determined by single particle electron cryomicroscopy to 10 A or below (till April 2015)

Protein Source Molecular

mass

(in MDa)

Medium Detector No. of

particlesaNo. of

asymmetric

unitsa

Resolution

(A)bEMDB Ref

Complex I Native (bovine) 1 Cymal-7 Falcon II 25,492 25,492 �5 2676 [58�]

V/A-ATPase Native (Thermus

thermophilus)

0.6 DDM Film 46,105 46,105 9 5335 [47�]

Native (Manduca

sexta)c0.9 C12E10 Falcon II 6714 6714 9.4 2781 [61�]

Native (S.cerevisiae) 0.9 DDM K2 50,030 50,030 6.9d 6284 [64�]

F1Fo-ATPase Native

(Polytomella sp.)

1.6 DDM Falcon II 37,238 74,476 �7 2852 [60�]

Glutamate

receptorcRecombinant 0.4 DDM Falcon II 21,360 42,720 7.6 2685 [57�]

Ryanodine

receptorcNative (rabbit

skeletal muscle)

2.2 Nanodisc Falcon II 25,000 100,000 6.1 2751 [38�]

Nanodisc TVIPS F816 94,354 377,416 8.5 2752 [38�]

CHAPS/lipids K2 46,400 185,600 4.8 6106 [39�]

Tween-20 Falcon II 65,872 263,488 3.8 2807 [37�]

DDM CCD 28,036 112,144 9.5 1275 [45]

CHAPS Film 25,722 102,888 10.2 5014 [46]

TRPV1 Recombinant 0.3 Amphipol A8-35 K2 37,310 149,240 3.3d 5778 [55��]

TRPA1 Recombinant 0.7 PMAL-C8 K2 20,733 82,932 �4d 6268 [63�]

g-Secretase Recombinant 0.17 Amphipol A8-35 K2 144,545 144,545 4.5 2677 [56�]

Digitonin K2 177,207 177,207 4.3 2974 [65]

Tmr AB+ AH5 Recombinant 0.18 DDM K2 102,000 102,000 8.2 6085 [59�]

Tmr AB Recombinant 0.135 DDM TVIPS F816 36,000 36,000 10 6087 [59�]

Anthrax prepore

toxin

Recombinant 0.44 NP-40 K2 60,455 423,185 2.9 6224 [62�]

Ribosome

complexese2.6–4.3

Sec61 Native (porcine) Digitonin Falcon II 80,019 80,019 3.35–3.9 2644, 46,

49, 50

[66]

Native (Canis sp.) Digitonin TVIPS F416 162,655 162,655 6.9 2510 [50]

Ssh1 Native (yeast), Ssh1(R) Digitonin Film 183,000 183,000 6.1 1651 [48]

SecYEG Native (E. coli) Nanodisc Film 85,664 85,664 7.1 1858 [49]

SecYEG (R)

SecYEb Native (M. jannaschii) DDM Film 37,000 37,000 9 5691 [53]

SecYEb (R)

Sec61+OST+

TRAP

Native (Triticum

or Canis sp.)

Digitonin TVIPS F416 15,705 15,705 9.3 2523 [52]

Digitonin Film 79,000 79,000 8.7 1528 [51]

YidC Native (E. coli)

YidC (R)

DDM TVIPS F416 58,960 58,960 8.0 2705 [54]

a Number of particles denotes the number used in the final map and number of asymmetric units is the total number averaged after the application of

symmetry.b The criteria used to estimate the resolution is the Fourier shell correlation (FSC) either at FSC at 0.143 or 0.5. The numbers denoted in bold are

reported at FSC 0.5.c The structure of V-ATPase from Manduca sexta, glutamate receptor and the early structures of ryanodine receptor (taken on film or CCD) have an

overall resolution of 10 A or better. However, the membrane domain is of lower resolutions and TM helices are not clearly resolved.d Multiple maps of V-type ATPase, TrpV1 and TrpA1 have been deposited and only the highest resolution is shown in the table. Three conformational

states of V-ATPase have been resolved and they are deposited in EMDB codes 6284–86. The structures of TrpV1 with ligands are deposited in EMDB

codes 5776–77 and please refer to Cao et al. 2013 for details of the structures [95�]. Similarly, the structures of TrpA1 with other ligands are deposited

in EMDB codes 6267 and 6269.e A collection of membrane proteins in complex with ribosomes are listed here. There are many more maps deposited in the database describing

different states and the list here is only a small collection chosen based on the species and describing only the highest resolution map. The resolution

shown is the overall resolution and in many of these maps, the membrane protein is of lower resolution, sometimes the TM-helices in the membrane

domain is not clearly resolved. The translocon proteins SSh1, SecYEG (E. coli) and SecYE (M. jannaschii) and YidC are all recombinant (shown in

brackets as ‘R’), while the ribosomes used in the study are native.

Abbreviations used in the table: EMDB: electron microscopy data base; DDM: n-dodecyl-b-maltopyranoside; Cymal-7: 7-cyclohexyl-1-heptyl-b-

maltoside; PMAL-C8: poly(maleic anhydride-alt-1-decene) substituted with 3-(dimethylamino) propylamine; C12E10: polyoxyethylene(10) dodecy-

lether; NP-40: Nonidet P-40; OST: oligosaccharyl-transferase; TRPV1: transient receptor potential V1; TRPA1: known for its extensive ankyrin

repeats at the amino terminal domain; TRAP: translocon associated protein complex.

Current Opinion in Structural Biology 2015, 33:103–114 www.sciencedirect.com


membrane proteins are shown. They clearly reveal distinct

views, internal details such as the transmembrane (TM)

helices and the detergent/lipid belt around the membrane

domain. The quaternary structure, integrity and conforma-

tional heterogeneity of the proteins become prominent in

such class averages (e.g. the tetrameric arrangement of RyR

in Figure 1b). Analysis from specimen preparation to

imaging and 2D classification can be done in a relatively

short period and provides a good feedback about biochem-

istry before large-scale data collection and reconstruction is

attempted. Such screening of membrane proteins and their

complexes, either by negative staining or by cryoEM, has

been instrumental in identifying the right constructs, lead-

ing to better crystals and structures and to study underlying

structural heterogeneity in solution [40–42].

From blobs to high-resolution structuresIn the early days of cryoEM of membrane proteins, a large

number of reconstructions have been published but they

showed relatively featureless blobs. There was also the

separate problem that reconstructions of the same protein

often yielded different results and interpretation. Fur-

ther, the propensity to average and refine noise resulted in

claims of resolution that could not be justified by the

maps [43,44]. Therefore, there was general skepticism

about whether reliable, high-resolution maps could be

obtained by single particle cryoEM. The maps of the

ryanodine receptor [45,46] (the membrane domain in

these RyR maps were of lower resolution) and the A

type-ATPase from Thermus thermophilus reconstructed

from images taken on a CCD or film were the first

examples to show that sub-nanometer resolution of mem-

brane proteins by cryoEM was possible [47�]. Multiple

structures of membrane proteins (the translocon, YidC) in

complex with ribosomes have been described but the

resolutions of the membrane protein part in many of these

reconstructions are generally lower than ribosomes and

often the TM helices are not clearly resolved [48–54].

Nevertheless, they have provided important biological

insights into the mechanism of protein translocation.

With direct electron detectors, it is now possible to

routinely visualize membrane proteins and obtain medi-

um to high-resolution maps in a relatively short period

(Table 1 and Figures 2 and 3). Some of the recent

structures of membrane proteins determined by single

particle cryoEM include transporters, enzymes, ion chan-

nels and multi-subunit membrane protein complexes

[37�,38�,39�,55��,56�,57�,58�,59�,60�,61�,62�,63�,64�,65].

The molecular mass of these proteins varies between

0.13 and 2.2 MDa. They contain 12–78 TM helices and

many of them lack any symmetry making these structures

truly ground breaking. The structures of RyR and ribo-

some in complex with Sec61 clearly highlight the advan-

tage of the direct detectors over film or CCD with an

increase in the resolution of the reconstructions despite

using fewer particles [37�,38�,39�,66].


Many membrane proteins by nature are dynamic and can

exhibit multiple conformations in solution. For crystalli-

zation, such structural heterogeneity has been overcome

by stabilizing a particular conformation using mutagene-

sis, antibodies or inhibitors. In cryoEM, such structural

heterogeneity can be computationally sorted into distinct

classes using recently introduced maximum likelihood

based 3D classification, which results in maps that are less

noisy and far superior [67,68]. Many of the membrane

protein structures listed in Table 1 have greatly benefit-

ted from such 3D classification and I foresee that this is

one area in image processing where future improvements

in software will help immensely in obtaining higher-

resolution maps of membrane proteins in distinct con-

formations with less particles.

One of the key areas in cryoEM where there has been lot

of ambiguity is the correctness and the actual resolution of

the maps [69]. In the past few years, several tests have

been introduced to validate cryoEM maps. Of these, the

tilt-pair validation test is a very powerful technique,

where two images are taken from the same area at

different tilt angles; this then allows the orientation of

a given particle to be checked against a map that is

thought to represent the protein of interest. The hand-

edness of the map can also be determined with this

technique [17�,70]. CryoEM images are noisy and invari-

ably noise builds up during the 3D refinement. High-

resolution noise substitution or randomization of phases

beyond certain resolution is an elegant way to check over-

fitting of the data [71]. Recently introduced ‘gold-stan-

dard refinement’ aims at preventing such over-fitting

already during refinement [72,73]. As the data quality

has improved, the maps have become better and it has

become now standard to expect certain structural features

to be seen with a given claim of resolution. For instance,

maps at resolutions better than 10 A should clearly show

the separation of a-helices. Resolving b-strands at �4.5 A

and the appearance of side chain densities of larger amino

acids at resolutions beyond 4 A are the next landmarks in

judging the resolution of the maps. It is also becoming

clear that the resolution often varies across the macro-

molecule with the core regions of the protein generally

show higher resolution than the peripheral regions of the

molecule. It is now common in cryoEM publications to

show a figure of the map depicting the local resolution

[74]. The gallery of membrane protein structures in

Figures 2 and 3 are in the resolution range of 3.3–9 A

and illustrate the structural features that one would

expect for each of these resolutions. As the resolutions

have started to reach 3 A or above, bound ions and ligands

are now being built confidently. Such model building and

refinement on cryoEM maps have benefitted from mod-

ifications of existing crystallographic tools [75–78].

For a biochemically well-characterized reasonably sized

membrane protein, with the present technology it is now


108 Membranes

Figure 2

(Nicastrinectodomain)

(A1)

(A0)

IN

OUT

γ-secretase(20 TM, 0.17 MDa, 4.5 Å)

TRPV1(24 TM, 0.3 MDa, 3.3Å)

A-ATPase(~32 TM, 0.6 MDa, 9Å)

(Fab)

ABC transporter(12 TM, 0.13 MDa, 8.2Å)


A gallery of membrane protein structures determined by single particle cryoEM. The maps have been selected to show a range of molecular

weight, complexity of the proteins and resolution. The heterodimeric ATP-binding cassette (ABC) transporter from Thermus thermophilus has

12 TM helices with an extended cytoplasmic ATP binding domain and has a molecular mass of �130 kDa. Such a small protein by itself can be

visualized and a �10 A map has been obtained [59�]. A complex of this ABC transporter with an Fab antibody fragment (50 kDa, shown in red)

that recognizes one of the subunit aids in more accurate orientation resulting in an 8 A map that clearly resolves the TM helices. g-Secretase is an

intramembrane aspartyl protease that cleaves a wide range of single pass TM substrates but widely known for cleaving amyloid b-peptide. Four

different subunits (PS, PEN-2, APH-1 and Nicastrin) are essential for assembly and functionality of the enzyme. The protein mass of g-secretase is

�170 kDa and the presence of the large ectodomain of Nicastrin greatly assists the alignment of the particles and a map at overall resolution of

4.5 A was obtained. The ectodomain is at higher resolution and much of the path of the polypeptide could be traced [56�]. The TM domain of g-

secretase is at lower resolution but sufficient to reveal the arrangement of TM helices, providing insights into how the substrate might approach

the interior of the enzyme. The transient receptor potential V1 (TRPV1) ion channel is a receptor for capsaicin and belongs to the family of ion

channels that are involved in sensing and transducing temperature [55��]. The structure of TRPV1 at 3.3 A is one of the highest resolution maps of

a membrane protein determined by single particle cryoEM. The map shows the classical fold of the voltage-gated ion channels with 6TM helices

per monomer in a tetrameric arrangement. Such a high-resolution map allowed de novo model building of almost the entire polypeptide chain

[55��]. Multiple structures of TRPV1 in complex with inhibitors have been obtained providing insights into the possible gating mechanism and

illustrate the power of cryoEM [95�]. The H+-driven A-type ATPase from Thermus thermophilus is a moderate sized membrane protein and the

reconstruction was performed with images captured on film. Though of lower resolution (�9 A), the structure shows the global architecture of the

A type-ATPases with the A1 catalytic domain, the Ao membrane domain, the central and the two peripheral stalks [47�]. The F and V-type ATPases

have equivalent F1/V1 and Fo/Vo domains that perform catalysis and ion translocation [60�,64�]. The horizontal helices in the Ao membrane domain

that we now know to exist from higher resolution maps of the F and V-type ATPases [60�,64�] can now be correlated in this low-resolution map.

routinely possible to get maps below 10 A with very little

effort. Depending on size, symmetry and sample hetero-

geneity, achieving higher resolutions may take a substan-

tial amount of time. The overall B-factor of the map, which

reflects the quality of the images and orientation accuracy,

is a useful indicator on how well the refinement is progres-

sing and if the imaging conditions are optimal. In particular,

it provides an estimate to the additional number of particles

required to reach a particular resolution [17�]. Thus, it is

advisable to set intermediate goals and it may be useful to

improve the biochemistry, specimen preparation and im-

aging rather than averaging millions of particles to find that


the resolution of the reconstruction increases only margin-

ally. Even at medium resolutions (such as 5–7 A) biological

insights can be inferred. Such examples from recent

structures include the description of the arrangement of

TM helices and the position of the ectodomain in g-

secretase, the identification of horizontal TM helices in

ATPase’s and the assignment and location of subunits in

bovine mitochondrial Complex I (Figures 2 and 3)

[56�,58�,60�,64�]. In the end resolution is just a number.

If a biological question can be addressed with a medium

resolution map then perhaps time can be better spent on

other problems.



Figure 3

B14.7

B16.6PGIV

(b)

(13 kDa)

Inner membrane

Matrix

(18 kDa)

42 kDa

B8

SDAP-α

SDAP-β

(MWFE+ B9)

B14(B13)

(a)

(c)


Architecture of mammalian respiratory Complex I. In the inner membrane of mitochondria (and the inner membrane of prokaryotes), the first step

in electron transfer is the oxidation of NADH by a multi subunit complex called NADH:Ubiqunione oxidoreductase or commonly called Complex I.

The electrons from NADH are transferred to ubiquinone through a series of iron-sulfur (FES) clusters and this transfer is coupled to proton

translocation across the membrane. The core subunits as defined in prokaryotes harbor all the catalytic subunits for electron transfer and proton

translocation [96]. In higher eukaryotes, the core subunits are augmented with varying numbers of accessory subunits that are involved in

assembly and regulation and are called supernumerary subunits. The bovine Complex I is a biochemically well studied enzyme with a molecular

mass of �1 MDa and is composed of >40 subunits [58�]. (a) A reconstruction of bovine Complex I at �5 A was obtained by cryoEM imaging of

the enzyme in detergent micelles. Two maps are overlaid to show two distinct feature of the enzyme. The eight FES clusters (shown in red) are

visible as the highest peaks in the map and the density for protein (gray) at intermediate threshold shows a number of TM helices spanning across

the membrane domain. For clarity, the detergent/lipid belt that is visible at lower threshold is not shown but the black bars mark the boundary

defined by them. (b) Assignment of subunits to Complex I. The map of Complex I has a nominal resolution of �5 A and some of the core regions

in particular TM helices are better resolved and start revealing the bulky side chains. The 14 core of subunits of bovine Complex I share high

homology with the prokaryotes. Using the atomic model of the Thermus thermophilus enzyme, the backbone and well resolved regions were built

[96]. For the assignment of supernumerary accessory subunits, a combination of biochemical and genetic information, secondary structure

prediction and subunits with known structures were employed. The identities of 14 supernumerary subunits have been assigned. These include

subunits that are membrane embedded as well as soluble proteins. The assigned subunits are colored in red and green and labeled with the

same color. The identities of the subunits whose names are labeled with brackets are less certain. Structural elements including several of the

supernumerary membrane subunits with a single TM helix have not been assigned (blue) in the current map. Figure reproduced with permission

from [58�]. (c) A view from matrix of mitochondria showing the arrangement of the TM helices in the membrane domain. The seven core

membrane subunit TM helices are shown in blue and the TM helices of supernumerary subunits in red. In total, bovine Complex I has 78 TM

helices. The curved nature of the membrane domain and the long horizontal helix are clearly visible. The detergent/lipid belt observed in the

cryoEM map is colored in green.

Present limits and possibilities of singleparticle cryoEMA question that is often asked is the lower limit in the size

of the protein for which a structure can be obtained by


single particle cryoEM. According to theory, structures of

proteins as small as hemoglobin (�64 kDa) can be

obtained [16��]. While such small proteins can be visual-

ized in micrographs, they cannot be oriented reliably with


110 Membranes

Figure 4

>10º

low

0.2º

high Orientation accuracy

Large

Viruses

Ribosomes(2-4 MDa)

ComplexI(~1 MDa)

(0.4 MDa )

γsec(0.17 MDa)

ABC(0.13 MDa)

TRPV1(0.3 MDa)

β-gal

(>2 MDa)

SmallMolecular weight (MDa)

0.5 Difficulty 10

BSA(0.07 MDa)


Structure determination of macromolecules by single particle electron

cryomicroscopy: present limits and possibilities. With the present

technology that includes microscopes with better vacuum, stable

stage, coherent beam, direct electron detector, software and

computing we can now achieve high-resolutions of macromolecules

by single particle cryoEM. Although, the theory says that structure

determination of macromolecules can be done with less particles and

smaller proteins, currently there are limits to what is possible [16��].

As explained in the introduction of the main text, the quality of the

map/reconstruction for a macromolecule depends on how accurately

it can be oriented. Large symmetrical molecules such as viruses can

be oriented accurately between 0.2 and 0.5 degrees and maps below

4 A can now routinely be obtained with around 1000 particles (due to

symmetry the number of asymmetric units averaged will be 60,000).

Ribosomes with their bound RNA have higher contrast and are less

sensitive to radiation damage. They have been one of the test

specimens in the development of single particle EM. The resolution of

ribosome maps has been gradually increasing and multiple

reconstructions with many different factors now dominate the

electron microscopy data bank (EMDB). Thus, in terms of difficulty

high-resolution structures of large symmetrical molecules and high

contrast objects such as ribosomes can be obtained with relative

ease. As the size of the macromolecule becomes smaller, it is

generally more difficult to obtain high-resolution structures and

requires a lot more effort. Small protein molecules such as

hemoglobin or bovine serum albumin (BSA) can be visualized in

micrographs but presently cannot be oriented accurately. Recent

structures of g-secretase (170 kDa) and ABC transporter

(130 kDa + 50 kDa Fab fragment) are highlighted in red to show the

smallest asymmetric structures determined to sub-nanometer

resolution by cryoEM at the moment [56�,59�]. The number of

particles required for a given protein to reach a resolution beyond 4 A


the present technology. Figure 4 tries to summarize the

current possibilities with cryoEM for macromolecules in

general. Large symmetric molecules such as viruses can

be oriented with high accuracy and at present a resolution

of 4 A or above can be obtained by averaging only few

thousand particles requiring less than half day of data

collection. As the size of the molecule decreases the

difficulty in obtaining such high-resolution structures

increases and often require averaging of 104–106 of asym-

metric units. Dynamic and structurally heterogeneous

membrane proteins such as the rotary ATPase’s may

require much more particles to classify distinct conforma-

tions and may still only give lower resolution maps

[60�,64�].

The structure of the 130 kDa heterodimeric ATP-bind-

ing cassette (ABC) transporter from T. thermophilus is to

date the smallest asymmetric protein structure that has

been determined to below 10 A by single particle cryoEM

[59�]. This was possible by the addition of a 50 kDa Fab

fragment, which binds to one of the monomer thus not

only increasing the size of molecule but greatly aiding the

orientation determination [59�]. Similarly, in the multi

subunit complex of 170 kDa g-secretase, the presence of

a large ectodomain helps in alignment of the molecule

[56�]. For small membrane proteins that may look like

spherical blobs (in particular if the membrane part is

covered with a large amount of detergent) it is possible

that addition of Fab fragment or small domains may

become a generic technique to obtain accurate orienta-

tion [79]. These examples represent what is currently

possible with cryoEM and highlight that not only the size

but also the shape matters for obtaining high-resolution

maps by single particle EM.

Conclusions and outlookRecent progress in the field of cryoEM has been aptly

called a ‘resolution revolution’ [80]. Some of the recent

examples show that it is possible to reach beyond 3 A

[62�,81–83]. While such resolutions are remarkable, it

does involve averaging a large number of particles, in

the range of 105–106 asymmetric units (Table 1). From

theory it is clear that similar resolutions should be

obtained with fewer particles [16��]. Analysis of the movie

frames from direct electron detectors reveals that the first

few frames are worse and have much less information

than expected [24,84]. This is one of the major reasons

why the images are still not perfect and why the averaging

of so many particles is still required. In current refinement

strategies, this is overcome either by discarding those

will depend on various factors but will largely be determined by its

stability and heterogeneity and one could expect to average 104–106

asymmetric units to achieve a resolution that can resolve side chains

for a wide range of molecules.



frames [23,82] or by down-weighting them [24,84]. Look-

ing ahead, the next technical advances in cryoEM will

come from two fronts. Firstly, understanding and curing

the problems associated with the initial frames and sec-

ondly, the next generation of direct electron detectors

with even higher DQE.

With respect to membrane proteins, won’t it be wonder-

ful if it becomes possible to visualize and obtain struc-

tures of membrane proteins in situ in their native

environment? For instance, ion channels at the synapse.

The technique of electron cryotomography, which has

been developed for a very long time, has yielded stunning

images of whole cells and thin sections [85]. With the

direct electron detectors and sub-tomogram averaging, it

is now possible to obtain structures of macromolecules in

their native environment, sometimes to sub-nanometer

resolution [86,87]. Recent analysis of the ATPase dimers

in the inner mitochondrial membrane, membrane associ-

ated mitochondrial ribosomes and the nicotinic acetyl-

choline receptor with rapsyn on the post-synaptic

membranes of the electric organ of Torpedo highlight

such possibilities for membrane proteins [88–90]. Two

recent advances namely the gold supported grids that

minimize some beam-induced movement [91�,92�] and

the phase plate [93,94] have great potential in acquiring

better images by tomography and increased resolution by

sub-tomogram averaging.

The future of cryoEM looks very promising and once a

few technical problems are overcome, in the near future it

may be possible to collect multiple data sets in a single

day just like the present day data collection from crystals

at synchrotrons.

Conflict of interestNothing declared.

Acknowledgements

I thank Xiaochen Bai and Rouslan Efremov for providing the micrographsand class averages of g-secretase and ryanodine receptor shown inFigure 1. I thank Richard Henderson, Nigel Unwin and Greg McMullan forcritical reading and comments on the manuscript. This work was supportedby the Medical Research Council Grant Number U105184322.

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http://dx.doi.org/10.1038/nmeth.3287
























































114 Membranes

94. Danev R, Buijsse B, Khoshouei M, Plitzko JM, Baumeister W: Voltapotential phase plate for in-focus phase contrast transmissionelectron microscopy. Proc Natl Acad Sci U S A 2014, 111:15635-15640.

95.�

Cao E, Liao M, Cheng Y, Julius D: TRPV1 structures in distinctconformations reveal activation mechanisms. Nature 2013,504:113-118.


A companion article to Ref. [55��] describing structures of TRPV1 channelin different states highlighting the power of cryoEM.

96. Baradaran R, Berrisford JM, Minhas GS, Sazanov LA: Crystalstructure of the entire respiratory complex I. Nature 2013,494:443-448.












membrane protein structures without crystals, by single ... · protein structures without crystals,...

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