Structure

Ways & Means

Benchmarking Membrane Protein Detergent

Stability for Improving Throughput
of High-Resolution X-ray Structures
Yo Sonoda,1,2,4,5 Simon Newstead,1,4,6 Nien-Jen Hu,1,2 Yilmaz Alguel,1,2,3 Emmanuel Nji,1,7 Konstantinos Beis,2
Shoko Yashiro,2,3 Chiara Lee,1 James Leung,1 Alexander D. Cameron,2,3 Bernadette Byrne,1 So Iwata,1,2,3,*
and David Drew1,*
1Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK
2Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton, Oxfordshire OX11 ODE, UK
3Japan Science and Technology Agency, ERATO, Human Receptor Crystallography Project, Yoshida Konoe, Sakyo-ku,

Kyoto 606-8501, Japan

4These authors contributed equally to this work
5Present address: Drug Discovery Research Department, Central Research Laboratories, Kaken Pharmaceutical Co. Ltd,

14 Shinomiya-minamigawara, Yamashina, Kyoto 607-8042, Japan

6Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
7Present address: School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland

*Correspondence: s.iwata@imperial.ac.uk (S.I.), d.drew@imperial.ac.uk (D.D.)

DOI 10.1016/j.str.2010.12.001
Open access under CC BY license.

SUMMARY solved (Aller et al., 2009; Pebay-Peyroula et al., 2003). These

Obtaining well-ordered crystals is a major hurdle to
X-ray structure determination of membrane proteins.
To facilitate crystal optimization, we investigated the
detergent stability of 24 eukaryotic and prokaryotic
membrane proteins, predominantly transporters,
using a fluorescent-based unfolding assay. We have
benchmarked the stability required for crystallization
in small micelle detergents, as they are statistically
more likely to lead to high-resolution structures.
Using this information, we have been able to obtain
well-diffracting crystals for a number of sodium and
proton-dependent transporters. By including in the
analysis seven membrane proteins for which struc-
tures are already known, AmtB, GlpG, Mhp1, GlpT,
EmrD, NhaA, and LacY, it was further possible to
demonstrate an overall trend between protein
stability and structural resolution. We suggest that
by monitoring membrane protein stability with refer-
ence to the benchmarks described here, greater
efforts can be placed on constructs and conditions
more likely to yield high-resolution structures.
INTRODUCTION
Structural determination of integral membrane proteins repre-
sents a major challenge. Integral membrane proteins make up
more than a quarter of all sequenced genomes (Wallin and von
Heijne, 1998) and yet, to date, only around 180 unique polytopic
structures have been elucidated (http://blanco.biomol.uci.edu/).
Transport proteins make up a large fraction of the membrane
proteome, and although numerous studies have shown that
transporters play key roles in drug pharamacokinetics (Giaco-
mini et al., 2010), only two mammalian structures have been
low transporter numbers are a reflection of the technical difficul-
ties of working with membrane proteins. As one might expect,
tools that can reduce the time it takes to solve novel membrane
protein crystal structures are in great demand.
C-terminal tagging of membrane proteins with green fluores-
cent protein (GFP) facilitates monitoring of both expression and
purification of target proteins (Drew et al., 2001, 2005). GFP fluo-
rescence from whole-cell culture and from SDS-polyacrylamide
gels provides a direct measure of the amount of membrane-inte-
grated expression (Drew et al., 2006) while ‘‘monodispersity’’
screening, by fluorescence-detection size-exclusion chromatog-
raphy (FSEC), makes it possible to evaluate the quality of the
expressed fusion using crude detergent solubilized extracts prior
to purification (Kawate and Gouaux, 2006). Selected homologs
can be grown for large-scale culturing and purification by
Ni2+-affinity chromatography by including a His6-10-tag on GFP.
Subsequent cleavage with a site-specific protease to remove
the GFP-His6-10 fusion, followed by a second step of Ni2+-affinity
chromatography, allows isolation of the untagged protein (Drew
et al., 2001, 2005). For these reasons, there is an increasing
number of membrane protein structures solved using material ex-
pressed and purified with a protease cleavable GFP-His6-10 fusion
tag: zebrafish ATP-gated P2X(4) receptor (Kawate et al., 2009),
chicken acid-sensing ion-channel (ASIC) (Jasti et al., 2007),
bacterial proton-coupled broad-specificity amino acid transporter
ApcT (Shaffer et al., 2009), rat ionotropic AMPA-glutamate
receptor GluA2 (Sobolevsky et al., 2009), chicken strong
inward-rectifier potassium channel Kir2.2 (Tao et al., 2009), bacte-
rial carnitine transporter CaiT (Tang et al., 2010), and red algae ClC
transporter CmCLC (Feng et al., 2010).
Although pipelines such as the one described here can greatly
facilitate overexpression and purification screening, arguably the
biggest obstacle to structure determination is crystal optimiza-
tion. It is generally thought that the membrane proteins that
remain stable in different detergent solutions are more likely to
form better ordered crystals. However, the effect of membrane

Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved 17

protein stability on crystal growth has not been systematically
studied.
To provide some quantitative measures we have determined
the stability of 17 pro- and eukaryotic transport proteins using
a fluorescent-based unfolding assay. We have analyzed this
data in relation to the quality of crystals obtained, defined by
how well crystals could be optimized to diffract X-rays to high-
resolution. As additional controls, seven membrane proteins
whose structures are already known were also included in
this analysis. Our findings show that that there is indeed a measur-
able correlation between the stability of the membrane protein in
detergent solution and the likelihood of obtaining well-ordered
crystals. Using this information, we have been able to obtain
X-ray structures for a number of secondary active transporters.
RESULTS AND DISCUSSION
GFP-Based Pipeline Strategy for Target Selection,
Purification, and Crystallization of Eukaryotic and
Prokaryotic Transporters
Previous screening of eukaryotic membrane proteins in Saccharo-
myces cerevisiae shows that only a quarter can be overexpressed
to levels suitable for structural studies (Newstead et al., 2007). For
this reason, we started by cloning a large number of eukaryotic
transporters
140, taken from 12 different transporter families
using cDNA from Oryza sativia, Arabadopsis thaliana, Mus
musculus, Rattus novergicus, or Homo sapiens, into the GFP-His8
containing 2m vector pDDGFP-2. The cloned targets were
screened in S. cerevisiae for full-length expression greater than
1 mg.l-1 using whole-cell and in-gel fluorescence, as described
previously (Newstead et al., 2007). Using the same criterion, we
cloned and screened 40 bacterial transporters in Escherichai
coli, of which most are homologs of those screened in S. cerevi-
siae, plus five archeal ABC transporters and six antibiotic resis-
tance proteins that share no eukaryotic sequence homology
(Drew et al., 2006). Bacterial and eukaryotic transporters that
displayed a sharp symmetrical FSEC elution profile in dodecyl-b-
D-maltopyranoside (12M) were subsequently grown in large-scale
cultures for purification and crystallization trials. As summarized in
Table 1A, we selected 47 transporters based on overexpression,
in-gel fluorescence and FSEC profiles for submission to purifica-
tion and crystallization trials.
In total, 17 of the 47 pro- and eukaryotic transporters purified
could be crystallized: 8 bacterial transporters (BT-1 to BT-8),
1 archeal (AT-1) and 8 eukaryotic transport proteins (PT-1 to
PT-4 and MT-1 to MT-4) (Figure 1; see Figures S1A and S1B
available online). An average crystallization success rate of

35% for both bacterial and eukaryotic transporters is compa-
rable to that obtained for globular proteins among structural
genomics consortia (Price et al., 2009).
The majority of transporter crystals diffracted X-rays between
8 and 10 Å, Table 1B. After standard crystallographic techniques
to try and improve crystal quality, only the eukaryotic transporter
PT-2 and the bacterial transporters BT-2 and BT-3 could be opti-
mized to diffract X-rays to 4 Å resolution or higher (Table 1B; Fig-
ure S2A). The best X-ray diffraction obtained by the other trans-
porter crystals was no greater than 5 Å. None of the mammalian
transporter crystals diffracted X-rays higher than 8 Å resolution
(MT1 to MT4).
18 Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Stability and Crystallization of Membrane Proteins
Crystal Optimization of Control Membrane Proteins
Although 12M has been the most successful crystallization
detergent for a-helical membrane proteins (Newstead et al.,
2008), it forms large micelles which can hinder crystal contacts
(Michel, 2001). To confirm that 12M is responsible for the poor
quality of the target transporter crystals, we repeated the crystal-
lization for seven other membrane proteins whose structures are
already known: LacY, lactose permease (Abramson et al., 2003);
AmtB, ammonium channel (Zheng et al., 2004); GlpG, rhomboid
protease (Wang et al., 2006); NhaA, sodium-proton exchanger
(Hunte et al., 2005); GlpT, glycerol-3-phosphate transporter
(Huang et al., 2003); EmrD, multidrug transporter (Yin et al.,
2006), and Mhp1, hydantoin transporter (Weyand et al., 2008).
To avoid biasing the analysis, we followed our standard purifica-
tion procedure outlined in Experimental Procedures for each of
these control proteins.
All our control proteins formed crystals in 12M using the
sparse-matrix screen MemGold (Figure S1C). As observed for
our target transporters, initial diffraction was also poor with
typical diffraction limits of
4–8 Å resolution (Table 1B). In
contrast, by repeating the purification of AmtB in n-dodecyl-
N,N-dimthylamine-N-oxide (LDAO), we obtained crystals with
a comparable quality to the reported structure, diffracting to
1.9 Å (Figure S2B). Similarly, Mhp1 crystals grown in n-nonyl-
b-D-maltopyranoside (9M) diffracted X-rays equivalent to those
published at 2.8 Å (Figure S2B). Although it proved impossible
to grow crystals of GlpG in nonyl-b-D-glucopyranoside (NG),
the protein we used for analysis is the full-length protein,
whereas the published structures are of the core protein lack-
ing 87 amino acids from the N terminus (Wang et al., 2006).
Nonetheless, after further trials, full-length GlpG crystals were
grown in n-decyl-b-D-maltopyranoside (10M) that diffracted
anisotropically to
3.5 Å (Figure S2B). These results strongly
suggest that using the detergent 12M is the main reason for
poor crystal quality. However, to confirm that protein produced
in our pipeline does not adversely affect crystal quality, crystals
for one of the control proteins (NhaA) were also optimized
in 12M.
After ten purifications from 5 liter cell-culture preparations,
NhaA crystals could be obtained that diffract X-rays to 3.5 Å
resolution in the best direction (Figure S2B). The arrangement
of NhaA in these crystals represents the physiological dimer, in
contrast to the nonphysiological monomeric X-ray structure
(Hunte et al., 2005; unpublished data). This side-by-side
arrangement of NhaA is consistent with that obtained by EM
and ESR (Appel et al., 2009; Hilger et al., 2007). In addition to
NhaA, we have also compared the crystallization of a bacterial
transporter (oligopeptide transporter, PepTSo) as a C-terminal
His6-tagged construct and as an untagged construct from the
GFP pipeline (Newstead et al., 2010). While the PepTSo-His6
crystals do not diffract X-rays beyond 4–5 Å, even after extensive
optimization, crystals grown using protein from our GFP-based
pipeline diffract to 3.6 Å resolution in the best direction (Figures
S1A and S2C). It is plausible that this difference is due to the
presence of the C-terminal His6 tag, which may hinder the
formation of well-ordered PepTSo crystals. The diffraction data
obtained from the untagged construct has enabled us to solve
the structure of PepTSo (Newstead et al., 2010); added to our
data set as BT-9 (Table 1B).
Structure
Stability and Crystallization of Membrane Proteins

Table 1. Summary of Crystallization and Corresponding X-ray Diffraction Obtained for Prokaryotic and Eukaryotic Transporters.
(A) The Summary of the Expression, Purification, and Crystallization Success Rates of the 140 Eukaryotic and 52 Prokaryotic Transporters
Screened from 12 Different Transporter Families

(B) Summary of the X-ray Diffraction Obtained from Transporter Crystals and Control Membrane Proteins Screened in (A)

Proteins are designated as BT, bacterial transport protein; AT, archeal transport protein; MT, mammalian transport protein; PT, plant transport protein.
Diffraction limits were binned into either >3.5, 4–8, or <8 Å due to anisotropic diffraction (>3.5 Å was restricted to crystals for which a complete data set
could be collected and scaled to this resolution or higher). Left brackets indicate initial resolution of screened crystals, no brackets indicates the reso-
lution obtained after further testing using standard crystallographic methods and right brackets, with resolution in italics, is the final resolution post-
stability analysis. All proteins were crystallized in 12M except the final resolution as noted for BT-5 (LDAO), AmtB (LDAO), GlpG (10M), and Mhp1 (9M).
The dashed line (-) means we were unable to discern clear X-ray diffraction. Note: diffraction shown here is for controls GlpT and GlpG is for the native
sequence; however, published structures contain and C- and N-terminal truncations respectively (Wang et al., 2003, 2006).

Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved 19

A B
Fluorescence
C
N
12 TMs 76 kDa
0 10 20 30 40 50 60 70 80 90 100
Fraction number
Figure 1. Example of the Experimental Data Measured for Each Membrane Protein Crystallized in the Detergent 12M
(A) Topology prediction of the rice anion exchanger (PT-2) with the position of the cysteines depicted as red spheres.
(B) FSEC trace of PT-2 in 12M-solubilized membranes.
(C) SEC trace of purified PT-2 and SDS-PAGE analysis of the pooled peak shown in the upper right panel; asterisk for PT-2 protein which migrates as two separate
bands in the gel as confirmed by mass-spectrometry.
(D) Crystal of PT-2 in the detergent 12M obtained after optimization (see Figures S1A and S1B for transporters BT1-9, MT1-4, and PT1-4; and Figure S2).
Benchmarking Membrane Protein Stability
in Detergent Solutions
In agreement with this analysis, statistically it has been shown
that a-helical membrane proteins that crystallize in 12M produce,
on average, lower resolution structures (Sonoda et al., 2010).
In particular, secondary-active transporters that lack large
hydrophilic domains tend to yield 3–4 Å resolution structures in
12M, which is insufficient if we are to fully understand mecha-
nistic details (Sonoda et al., 2010). Small micelle detergents are
an alternative to 12M; however, they represent a much harsher
environment for membrane proteins. Studies have suggested
that the chance of obtaining crystals in small detergents is
higher if the protein is more thermostable (Serrano-Vega et al.,
2008). The best documented example is stabilization of the
turkey b1-adrenergic receptor by alanine scanning mutagenesis
(Serrano-Vega et al., 2008). The thermostabilized receptor, with
a melting temperature 21 C higher than the native sequence,
crystallized in the short-chain detergent octylthioglucoside. The
GPCR structure in this detergent was solved to 2.7 Å resolution
(Warne et al., 2008).
One critical question is how stable does a given membrane
protein have to be in a small sized detergent to crystallize? To
address this conundrum, we measured the stability of the
target transporters and control proteins in six of the most
successful crystallization detergents using a fluorescent-based
unfolding assay (Alexandrov et al., 2008). In this assay the dye
N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]-maleimide
(CPM) principally becomes fluorescent upon reacting with free
sulfhydryl groups. As most cysteines are predominantly located
within TM segments, cysteine accessibility is a good measure of
protein unfolding (Alexandrov et al., 2008). All transporters in our
sample set, apart from AT-1, BT-2 and BT-8, were predicted to
have one or more transmembrane cysteine(s) (Krogh et al.,
2001) (Figures S1A–S1C). The ABC transporter AT-1 was there-
fore omitted from this analysis, while for BT-2 and BT-8 single
cysteine point mutants were measured instead (these cysteine
mutants yield crystals that diffract X-rays comparable to wild-
type protein). Protein at
10 mg.ml-1 in 0.03% 12M was diluted
150-fold into a Tris-HCl buffer (pH 7.5) containing each of the
20 Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Stability and Crystallization of Membrane Proteins
C D
MW (kDa)
250
UV 148
98
*
64
50
* 36
22
volume
following detergents: 12M, 10M, 9M, LDAO, dodecyl nonaethy-
lene glycol ether (C12E9) or n-octyl-b-D-glucopyranside (OG)
(see Experimental Procedures for further details). After incuba-
tion in the detergent buffer for 5 min, we monitored unfolding
using a 96-well spectrofluoremeter for 130 min at a single
temperature of 40 C (Figure S3). By calculating the fraction of
folded protein at each time point, we fitted a single exponential
decay curve (Figure 2A) as outlined previously (Roth et al.,
2008). Typically the highest CPM fluorescence over this time
period, taken as the ‘‘maximal’’ unfolded state, was observed
in the detergent LDAO.
As shown in Figure 2B, the mean stability of the bacterial trans-
porters, with a half-life (t1/2) = 93 min, is longest in the detergents
12M (72 kDa) and C12E9 (83 kDa) with the largest micelle sizes
(Strop and Brunger, 2005). The t1/2 is shorter as the micelle-
size of the detergents decreases, 10M (39 kDa) t1/2 = 54 min,
9M (31 kDa) t1/2 = 45 min, OG (26 kDa) t1/2 = 27 min and LDAO
(17 kDa) t1/2 = 18 min (le Maire et al., 2000; Bamber et al.,
2007; Strop and Brunger, 2005). Indeed, the mean unfolding
rates of the bacterial transporters correlate linearly with the
micelle size of the detergents, R2 = 0.95 (Figure S4). Strikingly,
the mean stability of the eukaryotic transporters is several-fold
lower than their bacterial counterparts in these detergents,
12M t1/2 = 21 min, C12E9 t1/2 = 18 min, 10M t1/2 = 19 min, 9M
t1/2 = 18, OG t1/2 = 12 min and LDAO t1/2 = 8 (Figure 2B).
However, because BT-1, BT-8, BT-3, MT-1, and PT-4 trans-
porters visually precipitated in OG we did not include their un-
folding values in this analysis. This may have resulted in some
bias, and because this observation adds some uncertainly as
to the validity of the other measurements in OG we decided
not to use the data from this detergent in any further analysis.
To verify the reliability of the t1/2 estimates measured in the other
detergents, we compared the unfolding rates in LDAO to the
FSEC profiles in this detergent. LDAO was selected as it is the
harshest of the six detergents screened.
As shown in Figure 3A and Figure S5, we can distinguish
a noticeable difference in the quality of the FSEC traces as the
t1/2 in LDAO decreases. All transporters and control membrane
proteins with a t1/2 longer than 15 min are equally monodisperse
 Structure
Stability and Crystallization of Membrane Proteins

A Figure 2. Membrane Protein Stability as Quantified

1.0 by the CPM Assay
(A) Example of the unfolding curves measured for each
membrane protein at 40 C for 130 min shown here
0.8
for the bacterial transporter BT-4 in the following deter-
gents: 12M, filled circle; 10M, filled triangle; 9M, filled
12M
Fraction of folded protein

diamond; LDAO, asterisk; C12E9, open triangle; OG, open

C12E9 circle.
0.6 (B) Mean unfolding rates for n = 16 bacterial (left panel) and
10M
9M n = 8 eukaryotic membrane proteins (right panel) in all
detergents C12E9, 12M, 10M, 9M, LDAO except OG
OG
0.4 where n = 12 and 6, respectively. The unfolding half-life
LDAO for each protein was calculated by fitting the data from
the CPM assay to a single exponential decay function as
described previously (Roth et al., 2008). See also Figures
0.2 S3 and S4.

0.0
0 50 100 150
Time (min) (black bars) with an unfolding t1/2 time of
B
17 min or longer at 40 C can reliably be
150 150 exchanged into LDAO (Figure 3B). For the
Prokaryoitc membrane protein mean half-life (min)

Eukaryotic membrane protein mean half-life (min)

control proteins (gray bars), only AmtB, Mhp1,

and GlpG-tr, which can be crystallized in either
LDAO or 9M detergents, pass this benchmark.
A t1/2 of
17 min or longer at 40 C also fits
100 100
the stability data obtained for 12M, in which all
proteins crystallized (Figure 3C). That is, apart
from MT-2 and MT-1 with a t1/2 close to this
benchmark of 16 min, most proteins have a
50 50 t1/2 = 20 min or longer. The stability of proteins
in C12E9 is also comparable to that measured
in 12M; only MT-2, t1/2 = 10 min, is clearly
unstable in this detergent (Figure S6A). In addi-
tion to 12M, C12E9, and LDAO detergents, we
0 0
consistently find that proteins with a t1/2 less
M

9M
E
9

9M
E

M
9
AO

AO
G

G
12

10

10
12
12

O
12

than 15 min in 10M and 9M always aggregate

C

C
LD

LD

Detergent Detergent upon exchange from 12M into these detergents

(data not shown). Thus, we estimate that the
majority of bacterial transporters, with a t1/2 of
in LDAO as they are in 12M. In contrast, all transporters that have 17 min or longer at 40 C, are also sufficiently stable to be
a t1/2 shorter than 15 min have FSEC traces in LDAO that are exchanged from 12M into 10M (9/9) and 9M (6/9) (Figures S6B
noticeably broader than those in 12M, with increasing amounts and S6C). Half of the eukaryotic transporters tested are suitable
of aggregation as the t1/2 becomes progressively shorter. for exchange into 10M (5/8) or 9M (4/8) (Figures S6B and S6C).
To verify at what t1/2 the proteins tested here will aggregate Of the few eukaryotic transporters stable in 10M and 9M deter-
with complete exchange into LDAO, all proteins showing similar gents, all are plant transporters, while none are mammalian
12M and LDAO traces were retested using purified protein. Apart transporters. This analysis is also consistent with our greater
from the control with the shortest t1/2 of 15 min (NhaA), all trans- success at obtaining better diffracting crystals for plant trans-
porters and control membrane proteins with a t1/2 of 17 min or porters (Table 1B).
longer could be exchanged from 12M into LDAO using SEC To assess if sensitivity toward lipid loss or incompatibility
and concentrated above 10 mg.ml-1. As a further test, we could be the reason for the poor detergent stability of the
submitted the transporters exchanged into LDAO for crystalliza- mammalian transporters, we added the lipid mixture PC: PE:
tion trials. PG into the SEC buffer containing 12M as previously
Bacterial transporters BT-5 and BT-8 crystallized in LDAO. described (Long et al., 2005). MT-2 and MT-3 transporters
While crystals for BT-8 were poor, the crystals for BT-5 improved were both more stable in 12M with lipid addition, although
from 7 Å in 12M, to 2.8 Å and finally to 2.2 Å resolution in LDAO no improvement was seen for MT-1 and MT-4 (data not
(Figure S2C). By using crystals grown in this detergent, we have shown). For MT-2 which showed the most improved stability,
recently been able to determine the structure for this transporter with an increase in t1/2 from 16 to 58 min, crystals grew with
(unpublished data) (Table 1B). a different morphology and diffract X-rays at much higher
Based on this analysis, we estimate four bacterial trans- resolution: 6 Å in the presence of lipid compared to 15 Å
porters (open bars) and none of the eukaryotic transporters without (Figure S7).

Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved 21
 Structure
Stability and Crystallization of Membrane Proteins

A B
i. ii. iii.
AmtB Mhp1 GlpG 40 LDAO
t1/2 = 35 min t1/2 = 21 min t1/2 = 21 min
R.F.U (arbitrary units)

R.F.U (arbitrary units)

R.F.U (arbitrary units)

30

Half-life (min)
20

17 min
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
Fraction number Fraction number Fraction number

10
iv. v. vi.
NhaA GlpT EmrD
t1/2 = 15 min t1/2 = 12 min t1/2 = 10 min

0
R.F.U (arbitrary units)

R.F.U (arbitrary units)

R.F.U (arbitrary units)

AmtB
Mhp1

NhaA

EmrD

LacY
BT-8

BT-2
BT-5
BT-9
BT-3

PT-4
BT-1
GlpT
BT-7

BT-6
PT-1

PT-2
BT-4

PT-3
MT-1

MT-2
MT-3

MT-4
GlpG-tr
C
Membrane protein
150
12M
>>

0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Fraction number Fraction number Fraction number
100
vii.
Half-life (min)

LacY
t1/2 = 6 min
R.F.U (arbitrary units)

50

17 min

0
AmtB

Mhp1

NhaA
EmrD

LacY
BT-3
BT-9
BT-5
BT-7
BT-8

BT-1
BT-6

GlpT
BT-2

BT-4

PT-1

PT-2
PT-3
PT-4
MT-4

MT-2
MT-3
MT-1
GlpG-tr

0 10 20 30 40 50 60 70 80 90 100
Fraction number

Membrane protein

Figure 3. Benchmarking Membrane Protein Detergent Stability

(A) Control membrane proteins FSEC traces in LDAO (green) and 12M (black) which have been ordered from most stable (left) to least stable (right), as determined
by their unfolding half-life in LDAO.
(B) Bars represent the unfolding half-life for each protein in LDAO.
(C) Bars represent the unfolding half-life for each protein in 12M. Bacterial proteins, open bars; Bacterial protein controls, light grey bars; Eukaryotic proteins,
black bars. The > sign above BT-3, 9 bars indicate that the half-life stability, although consistent in both 12M and C12E9 detergents (see Figure S6A) was longer
than the time measured to assess stability. See also Figures S5–S7.

Membrane Protein Stability Is Predominantly Intrinsic So far, we have obtained well-diffracting crystals for BT-2,
Not Detergent Specific BT-5, and BT-9 (Table 1B). With the exception of BT-8, these
To establish if membrane proteins that are comparatively more are the only transporters stable in LDAO, Figure 3B. In light of
stable in one detergent are also more stable in other types of this observation we focused on improving the diffraction from
detergents, we compared the membrane protein unfolding rates BT-8. After careful optimization, BT-8 crystals were obtained
of the proteins included in this study against one another. that diffract up to 3.3 Å in 12M (Table 1B; Figure S2C). If we
As shown in Figure S8A, there is a reasonably good correlation compare the stability of all the peptide transporters screened
between the t1/2 in 9M and 10M (R2 = 0.76). A similar correlation here, namely, BT-6, BT-7, BT-8, and BT-9, it is clear that BT-8
is observed for the t1/2 of the membrane proteins in 12M and and BT-9 are more stable overall (Figure S9). We suggest that
C12E9 (R2 = 0.86) Figure S8B, and also for 9M compared to this approach can aid in the selection of orthologs best suited
LDAO (R2 = 0.76) (Figure 4A). However, there are a number of for structural studies. Even with unrelated transporters we see
transporters that do not conform to this trend. For example, a stability trend, only the bacterial transporters stable in LDAO
Mhp1 is more stable in 9M, t1/2 = 56 min, than what would expect (BT-2, BT-5, BT-8, BT-9) have yielded crystals that diffract X-
from the half-life in 12M, t1/2 = 43, presumably because it rays from medium to high resolution (Figure 3B).
particularly favors this detergent; indeed, 9M was used for crys- A large-scale structure analysis of globular proteins concluded
tallization of Mhp1 (Weyand et al., 2008). Overall, however, these that crystallization propensity is not substantially influenced
findings strongly indicate that membrane protein stability is by thermodynamic stability. The best correlation is the fre-
predominantly intrinsic rather than detergent specific. quency of well-ordered surface epitopes capable of mediating

22 Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Stability and Crystallization of Membrane Proteins

A LDAO/9M resolution. For the control proteins, this is also the case. This
140 R = 0.76 correlation appeared in all tested detergents but again is clearest
140 LDAO 120 * in the detergent LDAO (Figure 4B).

t1/2 9M (min)
100
9M 80
120 * 60 Conclusion
40 In total, 25 pro- and eukaryotic membrane proteins consisting of
100 20
0
channels, enzymes, and primary and secondary active trans-
0 10 20 30 40 porters were crystallized from protein expressed as GFP fusions
80 in E. coli or S. cerevisiae. To overcome the greatest stumbling
Half-life (min)

t1/2 LDAO (min)

block for structure determination, that of crystal optimization,
60 we have measured the stability of 24 membrane proteins in
various detergents using a fluorescent-based unfolding assay.
40 Our findings indicate that membrane proteins with an unfold-
ing rate longer than approximately 17 min at 40 C are sufficiently
20 stable for crystallization trials in that detergent. Because we find
that membrane protein stability is an intrinsic property, rather
0 than one conferred by the detergent, proteins stable in harsher
BT-8
AmtB
MhpI
GlpG-tr
BT-2
BT-5
BT-9
BT-3

BT-1

BT-7
EmrD
PT-1
BT-6
MT-1
PT-2

MT-2
PT-3
MT-3
GlpT
PT-4

BT-4
NhaA

LacY

MT-4
detergents, like LDAO, are likely to be more stable in a more
diverse range of other detergents. These proteins are not only
B Membrane protein
more likely to form crystals in 12M that diffract X-rays from 3 to
35 3.6 Å, but also to higher resolution in a small micelle-sized deter-
R =0.86 2 gents. Using this information, we have been able to identify
target homologs for crystallization trials to speed up structural
30
AmtB (LDAO) determination. We have also been able to confirm a relationship
between the monodispersity of a membrane protein in crude
25
LDAO solubilized membranes with its purified stability. This
half-life LDAO (min)

further validates the use of the FSEC method for rapidly

20 GlpG (OG)
15
10
5 (12M)
0 have large hydrophilic domains do not need to be as stable
1.6 2.1 2.6
Published resolution (Å)
Figure 4. Membrane Protein Stability Is Predominantly Intrinsic and
Is Related to Its Propensity to Form Well-Ordered Crystals
(A) Bars represent the unfolding half-life for each protein in 9M (filled) and
plotted against that measured in LDAO (nonfilled); unfolding rates for LDAO
were plotted from the highest to lowest (left to right). Inset is a linear curve indi-
cating the average stability difference between LDAO and NM. Asterisk for
protein BT-3 indicates that we considered this difference as an outlier and,
as such, was not included in calculation of the correlation coefficient as dis-
played in inset.
(B) Membrane protein stability, as judged by unfolding rates in the detergent
LDAO, correlates to the published resolution of control membrane proteins.
The detergent used for crystallization is listed in brackets besides each protein.
See also Figure S8.
protein-protein interactions (Price et al., 2009). In agreement with
this analysis we also find no correlation between the stability of
the membrane protein in 12M and the propensity of the protein
to crystallize. Transporters we have been unable to crystallize
are, on average, as stable as those that do crystallize (data not
shown). However, if membrane proteins can be crystallized in
12M, our findings indicate that the better the stability the greater
the likelihood of optimizing crystals to diffract X-rays to higher
Mhp1(9M)
screening stable homologs prior to purification.
These findings do not mean those membrane proteins that do
NhaA (12M)
not pass this stability benchmark cannot be optimized in 12M to
GlpT yield well-diffracting crystals over time, e.g., NhaA. However, we
(12M)
EmrD
(12M) suggest that membrane proteins that pass this benchmark opti-
LacY
mize faster because proteins that are this stable have a larger
‘‘crystallization space.’’ Most likely membrane proteins that
3.1 3.6 4.1 because extensive crystal contacts can already be formed in
large micelle-sized detergents. This is also true for scaffold
approaches that increase the surface area for crystal contacts
in a mild detergent, e.g., T4-lysozyme GPCR fusions (Rose-
nbaum et al., 2007) and/or more general membrane protein
FAB/Fv monoclonal fragment complexes (Dutzler et al., 2003).
Still, even in both these cases, we argue that crystal optimization
is likely to be more fruitful if efforts are placed on the homolog that
is the most stable. If no naturally stable variants are found, then
effort can be placed on the identification of mutants or ligands
that stabilize the protein. In this regard it is important for future
stabilization strategies, in particular of mammalian homologs,
that we have verified that stability is predominantly intrinsic as it
further rationalizes the general use of such approaches.
In summary, by using the stability benchmarks outlined here, it
is possible to identify targets and optimize constructs that are
more likely to yield well diffracting crystals; by doing so we can
increase the rate at which new high-resolution eukaryotic and
prokaryotic membrane protein structures are solved.
EXPERIMENTAL PROCEDURES
Yeast and E. coli Genetic Manipulations
Eukaryotic membrane proteins were amplified from their respective
cDNA (either obtained from collaborators or purchased from imaGenes

Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved 23

http://www.imagenes-bio.de/), and bacterial/archeal membrane proteins from
genomic DNA with exception of E. coli clones which were obtained from an
already constructed E. coli GFP-fusion library (Daley et al., 2005). Cloning
into S. cerevisiae primers contained a 18 bp gene specific region and
a 30 bp homologous region on the forward 50 -TCG ACG GAT TCT AGA ACT
AGT GGA TCC CCC-30 and reverse primer 50 -AAA TTG ACC TTG AAA
ATA TAA ATT TTC CCC-30 . For cloning into E. coli vector pWaldo GFPe,
primers contained a 21 bp gene specific region, and a 50 -GCGCCCTCGAG-30
overhang on the forward primer for XhoI digestion and a 50 -CGCGCGGA
ATCC-30 overhang on the reverse primer for EcoRI digestion (Drew et al.,
2006). PCR product for eukaryotic genes and SmaI linearized pDDGFP-2
(Newstead et al., 2007) were transformed into the FGY217 strain (MATa,
ura3-52, lys2D201, pep4D) (Kota et al., 2007). Transformants were selected
on -Ura plates and positive clones initially confirmed by colony PCR and/or
whole-cell GFP fluorescence. For cloning into E. coli XhoI/EcoRI digested
bacterial or archeal PCR products were transformed with cut vector into
cloning cells and selected on plates containing 50 mg/ml Kanamycin.
Overexpression and Purification of Membrane Proteins
Using GFP-Based E. coli and S. cerevisiae Pipelines
Detailed step-by-step protocols describe our GFP-based overexpression
and purification pipeline in E. coli (Drew et al., 2006) and in S. cerevisiae
(Drew et al., 2008). In brief, membrane protein-GFP-His8 fusions were
selected based on fluorescence counts from 10 or 1 ml cultures correspond-
ing to more than 1 mg per liter. Membranes were isolated from 2 liter
cultures, resuspended in 10 ml of buffer, and 0.2 ml was used for solubiliza-
tion in 1% v/v with either of the detergents C12E9, 12M, 10M, 9M, or LDAO
and analyzed for monodispersity by FSEC. Monodisperse fusions in 12M
were selected for large-scale culturing (10–15 liters) and purified by IMAC
in 1 3 PBS buffer containing 0.1% 12M as detailed in referenced protocols.
Fusions were cleaved overnight with equimolar His6-TEV protease during
dialysis into 3 liter of crystallization buffer containing 20 mM Tris-HCl
(pH 7.5), 0.1M NaCl, 0.03% 12M. Digested material was passed through a
5 ml His-Trap column (GE Healthcare), and the flow through containing the
target protein collected. Membrane protein was concentrated, and loaded
onto a Superdex 200 10/30 size-exclusion column at 0.4 ml/min in crystalli-
zation buffer in the presence or absence of a lipid mixture (3PC: 1PE: 1PG,
Avanti Polar Lipids). The monodisperse protein peak was collected and
concentrated with either 50K or preferably 100K MWCO (Millipore) concen-
trators to
8–20 mg/ml.
Generalized Crystallization and Data Collection Strategy
Screening was carried out using the targeted sparse-matrix MemGold,
MemSys, and MemStart screens (Molecular Dimensions Ltd) using the
Mosquito crystallization robot (TTP LabTech) in 96-well plates (MRC,
Germany). All screens were set up with 200 nl drop sizes and at 19 C and
4 C. Crystal optimization was carried out manually using hanging drop plates
in a 24-well setup using drop sizes of 1 ml and using 96-well detergent and
additive screens (Hampton, Aliso Veijo, CA). Diffraction screening and data
collection was carried out at various synchrotron beamlines at the Diamond
Light Source (IO2, IO3, IO4, I24) and the ESRF (ID14eh4, ID29, ID23eh1, and
ID23eh2). Data were processed and scaled using the HKL suite of programs
(Otwinowski and Minor, 1997).
96-Well Fluorescent Based CPM Thermostability Assay
The thermostability assay was carried out essentially as described by Stevens
and co-workers (Alexandrov et al., 2008; Roth et al., 2008). One microliter of
purified membrane protein at
10 mg/ml was added to 150 ml of buffer con-
taining 20 mM Tris-HCl (pH 7.5), 0.1 M NaCl, and detergent at three times
CMC in a 96-well black Nunc plate. CPM dye at 4 mg/ml in DMSO was diluted
100-fold into buffer containing 20 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.03%
DDM, and warmed to room temperature. Three microliters of dye at 40 mg/ml
was added, clear plate cover set in place, and within 5 min from protein addi-
tion fluorescence emission was measured at 463 nm (excitation 387 nm) on the
SpectraMax2e plate reader (Molecular Devices) at 40 C. Recordings were
measured every 5 min for 3 hr with 15 s shaking interval between each reading.
The fraction of folded protein at each time point was calculated by the quotient
of raw fluorescence measured at each time point divided by the maximal fluo-
24 Structure 19, 17–25, January 12, 2011 ª2011 Elsevier Ltd All rights reserved
Structure
Stability and Crystallization of Membrane Proteins
rescence measured for the detergent series. A single exponential decay curve
was plotted using GraphPad Prism software (San Diego, CA).
SUPPLEMENTAL INFORMATION
Supplemental Information includes nine figures and can be found with this
article online at doi:10.1016/j.str.2010.12.001.
ACKNOWLEDGMENTS
We thank Dan Daley for E. coli membrane protein GFP-fusion plasmids,
Simone Weyand, Peter Henderson, Sebastian Sauterm, and Novandy Lim
for advice or assistance with control proteins. We also thank Edmund Kunji,
Jan-Willem de Gier and Gunnar von Heijne and the reviewers for critical
reading of the manuscript and useful comments. Funded by the grants from
the UK BBSRC (BB/G023425/1 to S.I.) and Membrane Protein Structure Initia-
tive (MPSi) (grant BBS/B/14418 to S.I.), Wellcome Trust for funding of the
Membrane Protein Laboratory (WT089809/Z/09/Z to S.I.), the EU-PF6
E-MEP European Membrane Protein Consortium (to S.I.), the FP7 EDICT
project (210924 to B.B. and S.I.), and the Royal Society through the University
Research Fellowship scheme (to D.D.).
Received: September 17, 2010
Revised: November 30, 2010
Accepted: December 6, 2010
Published: January 11, 2011
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