research communications
Acta Cryst.
(2020). F
76
https://doi.org/10.1107/S2053230X20010572
1of8
Received 7 April 2020
Accepted 31 July 2020
Edited by G. G. Prive
́
, University of Toronto,
Canada
Keywords:
membrane-protein purification;
crystallization contaminant; transcription termi-
nation factor; cryoEM.
PDB reference
:
E. coli
transcription termination
factor Rho, 6wa8
Supporting information
:
this article has
supporting information at journals.iucr.org/f
Crystal structure of the
Escherichia coli
transcription termination factor Rho
Chengcheng Fan
a
and Douglas C. Rees
a,b
*
a
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard,
Pasadena, CA 91125, USA, and
b
Howard Hughes Medical Institute, California Institute of Technology, 1200 East
California Boulevard, Pasadena, CA 91125, USA. *Correspondence e-mail: dcrees@caltech.edu
During the crystal structure analysis of an ATP-binding cassette (ABC)
transporter overexpressed in
Escherichia coli
, a contaminant protein was
crystallized. The identity of the contaminant was revealed by mass spectrometry
to be the
Escherichia coli
transcription terminator factor Rho, structures of
which had been previously determined in different conformational states.
Although Rho was present at only
1% of the target protein (a bacterial
homolog of the eukaryotic ABC transporter of mitochondria from
Novo-
sphingobium aromaticivorans
;
Na
Atm1), it preferentially crystallized in space
group
C
2 as thin plates that diffracted to 3.30 A
̊
resolution. The structure of Rho
in this crystal form exhibits a hexameric open-ring staircase conformation with
bound ATP; this characteristic structure was also observed on electron-
microscopy grids of the
Na
Atm1 preparation.
1. Introduction
One of the challenges in the crystallography of membrane
proteins is their typically low expression level, which neces-
sitates a significant degree of purification to separate the
protein of interest from all other cellular proteins. This can
consequently lead to the inadvertent purification of contami-
nant proteins that might otherwise be present at negligible
levels when the target protein expresses at high levels. In
unfortunate cases, these impurities may crystallize more
readily than the target protein, leading to misplaced enthu-
siasm until the contaminant is recognized. As examples, the
multi-drug efflux pump AcrB is a well known crystallization
contaminant in membrane-protein preparations owing to its
relatively high expression level during recombinant protein
expressions with antibiotic selection and its nonspecific
binding to Ni–NTA columns (Veesler
et al.
, 2008; Das
et al.
,
2007). Bacterioferritin has also been crystallized as a contami-
nant in preparations of cytochrome
cbb
3
oxidase (Nam
et al.
,
2010). In addition, exogenous proteins such as DNase, lyso-
zyme and various proteases used in target protein purification
have also been shown to be crystallization contaminants
(Niedzialkowska
et al.
, 2016). Compilations facilitate the
identification of crystals of contaminant proteins (Hungler
et
al.
, 2016; Simpkin
et al.
, 2018), but the crystallization of ‘novel’
impurities is still a concern. In this work, we report the crystal
structure of a previously unreported contaminating protein,
the transcription termination factor Rho from
Escherichia
coli
, which was obtained during the structural analysis of a
bacterial ATP-binding cassette (ABC) transporter.
ISSN 2053-230X
Rho is a hexameric RNA helicase that functions in tran-
scription termination in
E. coli
. The six subunits together form
a ring-like structure, and the structure switches between an
open-ring staircase conformation and a closed-ring confor-
mation coupled to the binding and hydrolysis of ATP (Skor-
dalakes & Berger, 2003; Thomsen & Berger, 2009). Here, we
present the crystal structure of Rho in an open-ring staircase
conformation at 3.30 A
̊
resolution with ATP bound.
2. Materials and methods
2.1. Macromolecule production
Rho copurified with the bacterial ABC exporter
Na
Atm1,
which is a homolog of the ABC transporter of mitochondria
(Atm1) from
Novosphingobium aromaticivorans
, using an
E. coli
expression system and a modified version of a
previously published protocol (Lee
et al.
, 2014). Briefly, frozen
E. coli
cell pellets containing overexpressed
Na
Atm1 (and
Rho) were lysed in lysis buffer consisting of 100 m
M
NaCl,
20 m
M
Tris pH 7.5, 40 m
M
imidazole pH 7.5, 10 m
M
MgCl
2
,
0.5%(
w
/
v
)
n
-dodecyl-
-
d
-maltopyranoside (DDM; Anatrace),
0.5%(
w
/
v
) octaethylene glycol monododecyl ether (C12E8;
Anatrace) with the addition of lysozyme, DNase and protease
inhibitor. After stirring for 3 h at 4
C, the lysate was subjected
to ultracentrifugation at
113 000
g
for 45 min at 4
C. The
supernatant was collected, loaded onto a pre-washed Ni–NTA
column in buffer consisting of 100 m
M
NaCl, 20 m
M
Tris pH
7.5, 50 m
M
imidazole pH 7.5, 0.05% DDM, 0.05% C12E8 and
eluted with the same buffer containing 350 m
M
imidazole pH
7.5. The protein was further purified by size-exclusion chro-
matography (SEC) using a HiLoad 16/60 Superdex 200
column (GE Healthcare) with SEC buffer consisting of
100 m
M
NaCl, 20 m
M
Tris pH 7.5, 0.05% DDM, 0.05%
C12E8. Peak fractions were collected and concentrated to
20 mg ml
1
using a 100 kDa cutoff Amicon Ultra 15
concentrator (Millipore). Macromolecule-production infor-
mation is summarized in Table 1.
2.2. Crystallization
Rho crystallized during the crystallization trials of
Na
Atm1
under optimized conditions based on MemGold (Molecular
Dimensions) condition No. 68 at 20
C by hanging-drop vapor
diffusion. The final crystallization condition consisted of
100 m
M
NaCl, 100 m
M
Tris pH 8.3, 28% polyethylene glycol
2000 monomethyl ether (PEG 2000 MME), 0.2
M
non-
detergent sulfobetaine 221 (NDSB-221), 20 m
M
ATP pH 7.5.
The crystallization sample was prepared in 1 m
M
ATP pH 7.5,
5m
M
EDTA pH 7.5 in the presence and absence of 5 m
M
oxidized glutathione (GSSG) pH 7.5, which is a transport
ligand for
Na
Atm1. Thin plate-shaped crystals appeared in
about two weeks. The crystals were harvested in cryoprotec-
tant solution consisting of 100 m
M
NaCl, 100 m
M
Tris pH 8.3,
28% PEG 2000 MME with PEG 400 at 10%, 15% and 20%
before flash-cooling in liquid nitrogen. Crystallization infor-
mation is summarized in Table 2.
2.3. Data collection and processing
Crystals were screened on the GM/CA beamline 23-ID-B at
the Advanced Photon Source (APS) and on beamline 12-2 at
the Stanford Synchrotron Radiation Laboratory. The final
data set was collected on the GM/CA beamline 23-ID-B with
an EIGER 16M detector (Dectris) using
JBluIce
–
EPICS
(Stepanov
et al.
, 2011), processed and integrated with
XDS
(Kabsch, 2010) and scaled with
AIMLESS
(Evans &
Murshudov, 2013). The crystals of Rho diffracted to about
3.30 A
̊
resolution in space group
C
2, with unit-cell parameters
a
= 161.8,
b
= 101.9,
c
= 184.0 A
̊
,
= 107.8
. Data-collection
and processing statistics are summarized in Table 3.
2.4. Structure solution and refinement
The self-rotation function was calculated with the
CCP
4
program
MOLREP
(Winn
et al.
, 2011). Molecular replace-
ment was performed with
Phaser
in
Phenix
(Liebschner
et al.
,
2019) using a monomeric subunit of a previous structure of
Rho with PDB code 1pvo (Skordalakes & Berger, 2003) as a
model. Initial jelly-body refinements were carried out with
REFMAC
5in
CCP
4(Winn
et al.
, 2011). Subsequent iterative
refinement and model-building runs were separately
conducted with
phenix.refine
(Liebschner
et al.
, 2019) and
Coot
(Emsley
et al.
, 2010), respectively. The refined coordinates
research communications
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Fan & Rees
Transcription termination factor Rho
Acta Cryst.
(2020). F
76
Table 1
Macromolecule-production information.
The protein was expressed in
E. coli
from the native promotor without the use
of an expression plasmid.
Source organism
E. coli
DNA source
Genomic DNA from
E. coli
Expression host
E. coli
BL21-Gold (DE3)
Complete amino-acid sequence
of the construct produced
MNLTELKNTPVSELITLGENMGLENLARMR
KQDIIFAILKQHAKSGEDIFGDGVLEIL
QDGFGFLRSADSSYLAGPDDIYVSPSQI
RRFNLRTGDTISGKIRPPKEGERYFALL
KVNEVNFDKPENARNKILFENLTPLHAN
SRLRMERGNGSTEDLTARVLDLASPIGR
GQRGLIVAPPKAGKTMLLQNIAQSIAYN
HPDCVLMVLLIDERPEEVTEMQRLVKGE
VVASTFDEPASRHVQVAEMVIEKAKRLV
EHKKDVIILLDSITRLARAYNTVVPASG
KVLTGGVDANALHRPKRFFGAARNVEEG
GSLTIIATALIDTGSKMDEVIYEEFKGT
GNMELHLSRKIAEKRVFPAIDYNRSGTR
KEELLTTQEELQKMWILRKIIHPMGEID
AMEFLINKLAMTKTNDDFFEMMKRS
Table 2
Crystallization.
Method
Hanging-drop vapor diffusion
Plate type
Hampton Research VDX
Temperature (K)
293
Protein concentration (mg ml
1
)<1
Buffer composition of protein
solution
100 m
M
NaCl, 20 m
M
Tris pH 7.5,
1m
M
ATP
Composition of reservoir solution
100 m
M
NaCl, 100 m
M
Tris pH 8.3,
20 m
M
ATP, 200 m
M
NDSB-221,
28% PEG 2000 MME
Volume and ratio of drop
1
m
l protein solution + 1
m
l reservoir
solution
Volume of reservoir (
m
l)
500
and structure factors have been deposited in the RCSB
Protein Data Bank as entry 6wa8. Refinement statistics are
summarized in Table 4.
2.5. Electron-microscopy sample preparation and data
processing
The expression plasmid for the membrane-scaffolding
protein MSP1D1 was purchased from Addgene (plasmid No.
20061). The expression and purification of MSP1D1 were
carried out using published protocols with minor modifica-
tions (Ritchie
et al.
, 2009). Reconstitution of
Na
Atm1 (and
the Rho contaminant) with MSP1D1 was carried out with
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) at a
1:2:130 molar ratio of
Na
Atm1:MSP1D1:POPC. This recon-
stituted sample was incubated overnight at 4
C. After two
hours of incubation, BioBeads were added at 200 mg ml
1
for
detergent removal. The sample was then subjected to size-
exclusion chromatography on a Superdex 200 Increase 10/300
column (GE Healthcare). The peak fractions were pooled and
concentrated to
8mgml
1
.
EM grids were prepared using a protein concentration of
4mgml
1
in the presence of 5 m
M
GSSG and 5 m
M
AMPPNP. 3
m
l protein sample was applied onto freshly glow-
discharged QuantiFoil Cu R2/2 300 mesh grids and blotted
for 4 s with a blot force of 0 and 100% humidity at room
temperature using a Vitrobot Mark IV (FEI). Data were
collected on a 200 keV Talos Arctica with a Falcon III detector
at a magnification of 92 000 and a total dose of 81 e A
̊
2
at the
Caltech CryoEM Facility.
Data processing was performed with
cryoSPARC
2 (Punjani
et al.
, 2017), following motion correction with full-frame
motion and estimation of the contrast transfer function (CTF)
with
CTFFIND
(Rohou & Grigorieff, 2015). Particles were
picked using a reconstruction of
Na
Atm1 as a template and
extracted in
cryoSPARC
2. The initial 2D classification
revealed a single class of Rho with
2500 particles. The
particles were then 2D-classified again into five classes, with
four good classes with a total of
2200 particles, as shown in
Fig. 4(
b
).
3. Results and discussion
Crystals of Rho were unexpectedly obtained during studies of
the bacterial ABC transporter
Na
Atm1 from
N. aromatici-
vorans
.
Na
Atm1 was recombinantly expressed in
E. coli
BL21-
Gold (DE3) cells with a C-terminal 6
His tag following a
previously established protocol (Lee
et al.
, 2014). After solu-
bilization of the
E. coli
cells in DDM and C12E8, protein
purification proceeded by Ni–NTA affinity purification
followed by size-exclusion chromatography (Fig. 1
a
). SDS–
PAGE gels indicated a high degree of purity, although in
subsequent analysis of overloaded gels a small amount of
another protein was present at a molecular weight of
40 kDa
(Fig. 1
b
).
The crystals obtained during the crystallization optimiza-
tion belonged to space group
C
2, with unit-cell parameters
a
= 161.8,
b
= 101.9,
c
= 184.0 A
̊
,
= 107.8
. The asymmetric
unit volume was of sufficient size to accommodate an
Na
Atm1
dimer (molecular weight of 133 kDa). Analysis of the self-
rotation function calculated from the diffraction data revealed
a noncrystallographic symmetry (NCS) sixfold axis offset
3–4
from the crystallographic
a
axis. Interaction of the
perpendicular twofold and sixfold axes generates a set of
noncrystallographic twofold rotation operations separated by
60
in the plane perpendicular to the NCS sixfold axis (Fig. 1
c
).
Given the unit-cell dimensions, this apparent NCS was
incompatible with dimeric
Na
Atm1, which raised the possi-
bility that a contaminant had crystallized. With an estimated
molecular weight of
40 kDa based on the observation of a
research communications
Acta Cryst.
(2020). F
76
Fan & Rees
Transcription termination factor Rho
3of8
Table 3
Data collection and processing.
Values in parentheses are for the outer shell.
Diffraction source
GM/CA 23-ID-B, APS
Wavelength (A
̊
)
0.9793
Temperature (K)
100
Detector
EIGER 16M
Crystal-to-detector distance (mm)
400
Rotation range per image (
)
0.2
Total rotation range (
)
360
Exposure time per image (s)
0.2
Space group
C
2
a
,
b
,
c
(A
̊
)
161.75, 101.90, 184.02
,
,
(
)
90, 107.8, 90
Mosaicity (
)
0.11
Resolution range (A
̊
)
39.79–3.30 (3.42–3.30)
Total No. of reflections
304610 (29798)
No. of unique reflections
43031 (4465)
Completeness (%)
99.9 (99.7)
Multiplicity
7.1 (6.7)
h
I
/
(
I
)
i
6.6 (1.5)†
R
p.i.m.
0.103 (0.729)
Overall
B
factor from Wilson plot (A
̊
2
)
79.1‡
† Overall resolution cutoff determined by data completeness and CC
1/2
>0.50inthe
high-resolution shell.
I
/
(
I
) falls below 2.0 past 3.46 A
̊
resolution. ‡ There were ice
rings in the data.
Table 4
Structure solution and refinement.
Values in parentheses are for the outer shell.
Resolution range (A
̊
)
38.51–3.30 (3.42–3.30)
Completeness (%)
99.6 (97.3)
Cutoff
8.9 (1.3)
No. of reflections, working set
40807 (3928)
No. of reflections, test set
2095 (217)
Final
R
cryst
0.252 (0.320)
Final
R
free
0.296 (0.364)
No. of non-H atoms
Total
19776
Protein
19590
Ligand
186
R.m.s. deviations
Bond lengths (A
̊
)
0.002
Angles (
)
0.58
Average
B
factors (A
̊
2
)
Overall
101.8
Protein
101.4
Ligand
143.2
Ramachandran plot
Most favored (%)
95.5
Allowed (%)
4.2
PDB entry
6wa8
faint impurity band in the gel and a Matthews coefficient
analysis, we performed molecular replacements with known
crystallization contaminants (Hungler
et al.
, 2016; Simpkin
et
al.
, 2018), which all failed to yield a molecular-replacement
solution.
The identification of Rho (molecular weight 47 kDa) was
established by a mass-spectrometric analysis of the peptides
prepared by trypsin digestion of the protein in the SDS–PAGE
bands. Using this information, we were able to obtain a
molecular-replacement solution using Rho in the AMPPNP-
bound state (PDB entry 1pvo; Skordalakes & Berger, 2003) as
the input model. The molecular-replacement results estab-
lished that in these crystals Rho adopts a six-membered
broken-staircase conformation. The structure was refined to
an
R
work
and
R
free
of 25.2% and 29.6%, respectively (Table 4).
The electron-density map also revealed ATP to be bound in all
six subunits (Fig. 2
a
).
The individual Rho subunits are structurally similar overall,
except for the terminal subunit at the ‘break’ in the staircase,
where the first 50 residues at the N-terminus exhibited a shift
of 2–8 A
̊
relative to the other monomers (Fig. 2
b
). The r.m.s.d.
between the present structure and the molecular-replacement
input model (PDB entry 1pvo) is 2.5 A
̊
using the C
positions
for the superposition and 3.3 A
̊
when using all atoms of the six
subunits in the hexamer. The r.m.s.d.s between individual
subunits in the present structure and PDB entry 1pvo are
0.59–0.73 A
̊
, reflecting their similar tertiary structures. The
relationship between adjacent subunits in the broken staircase
of the present structure may be approximated by a screw
operation with a rotation around the screw axis of 60.5
per
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Fan & Rees
Transcription termination factor Rho
Acta Cryst.
(2020). F
76
Figure 1
Purification and self-rotation function analysis of Rho. (
a
) Size-exclusion chromatograph of
Na
Atm1 purification using a HiLoad Superdex 200 16/60
column. The column void volume is colored gray, while the elution positions of various molecular-weight standards (in kDa) are marked on the
chromatograph. (
b
) SDS–PAGE of the peak fractions of SEC purification from (
a
). (
c
)The
= 180
and 60
sections of the self-rotation function
calculated using
MOLREP
in
CCP
4(Winn
et al.
, 2011) with an integration radius of 51 A
̊
using diffraction data between 3.3 and 40 A
̊
resolution.
subunit and a corresonding translation along the axis of
7.7 A
̊
. In comparison, the corresponding values for PDB
entry 1pvo are 57.9
and
8.2 A
̊
, respectively. Reflecting the
larger rotation angle per subunit, the present structure exhi-
bits a more closed ring in comparison to the original broken-
staircase structure (Figs. 3
a
and 3
b
). At the level of subunit–
subunit interactions, however, the differences are subtle and
we could not identify the specific interactions responsible for
these differences in hexamer structure. Of note, the nucleotide-
binding site is at the interface between subunits and small
changes in subunit–subunit interactions may reflect the
presence of different nucleotides: ATP in this structure and
either no nucleotide (apo), ATP
S or AMPPNP in other
open-ring structures (Skordalakes
et al.
, 2005; Skordalakes &
Berger, 2003).
How did Rho end up in our crystallization conditions? Our
hypothesis is that during the overexpression of proteins the
E. coli
transcription and translation machineries are highly
expressed for the production of mRNAs and recombinant
proteins, respectively. Rho, as the termination factor, would
plausibly be overexpressed as part of the transcription-
termination process; thus, it is likely that Rho is a general
contaminant and does not arise specifically from the over-
expression of
Na
Atm1. The fact that Rho eluted from the Ni–
NTA column along with His-tagged
Na
Atm1 suggests that
there is nonspecific binding to the Ni
2+
resin by the histidine
residues distributed throughout the whole protein (Fig. 3
c
).
The open-ring conformation that Rho adopts in solution
(Thomsen
et al.
, 2016) with a molecular weight of 282 kDa for
the hexamer apparently has a comparable hydrodynamic
research communications
Acta Cryst.
(2020). F
76
Fan & Rees
Transcription termination factor Rho
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Figure 2
Crystal structure of Rho. (
a
) Overall view of Rho in broken-staircase conformation with ATP bound. (
b
) Single subunit of Rho with ATP bound. The six
subunits are shown separately and ATP is shown as red spheres. The dashed circle identifies the N-terminal
50 residues of the Rho subunit positioned
at the break in the hexameric staircase arrangement; this region has rearranged in this subunit relative to the conformation exhibited in the other fiv
e
subunits.
radius to
Na
Atm1, with a dimer molecular weight of 133 kDa
in addition to the detergent micelle. Given the low abundance,
the presence of Rho in the SEC fractions was only detected in
hindsight. Also in hindsight, Rho was not present in the
original
Na
Atm1 purification, which included a membrane-
isolation step in which Rho was presumably removed (Lee
et
al.
, 2014); in the present work the membrane-isolation step
was omitted and Rho subsequently copurified with
Na
Atm1.
We have also observed Rho in single-particle cryoEM
studies of
Na
Atm1 reconstituted in membrane-scaffolding
protein (MSP) nanodiscs. The
Na
Atm1 nanodisc sample was
prepared by incubating detergent-purified
Na
Atm1 with
MSPs and lipids, and was further purified by size-exclusion
chromatography. Similar to the purification in detergent, the
peak fractions were collected for single-particle cryoEM
sample preparation (Fig. 4
a
). The 2D classification reported
one class of Rho in the broken hexameric state (Fig. 4
b
), again
suggesting that Rho has a similar hydrodynamic radius and
plausibly a similar molecular weight to our reconstituted
Na
Atm1 in nanodiscs.
In a structural analysis of a prokaryotic chloride channel, a
single peptide of Rho was identified in a mass-spectrometric
analysis of the gel band (Abeyrathne & Grigorieff, 2017),
representing the first time, to our knowledge, that Rho has
been identified as a possible contaminant during membrane-
protein expression. As demonstrated in this report, Rho can
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Fan & Rees
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Figure 3
Overall architecture of Rho. Overall structural representations of (
a
) ATP-bound Rho (this study) and (
b
) the AMPPNP-bound structure of Rho (PDB
entry 1pvo; Skordalakes & Berger, 2003). (
c
) Distribution of surface histidine residues (blue) in the ATP-bound structure of Rho (this work). The
spacings between the surface-exposed histidines are several nanometres and are comparable to the loading density of His-tagged proteins bound to Ni
–
NTA beads (Hayworth & Hermanson, 2014), which presumably allows multiple binding sites to Ni–NTA and contributes to the observed affinity of Rho
for Ni–NTA resin.
crystallize even in the presence of a large excess of other
proteins, and thus it should be added to the list of known
contaminant proteins in crystallography.
Acknowledgements
We thank Jens T. Kaiser and the beamline staff at Advanced
Photon Source GM/CA beamline 23-ID-B and Stanford
Synchrotron Radiation Lightsource (SSRL) beamline 12-2 for
their support during crystallographic data collection, Mona
Shahgholi at the Caltech CCE Multiuser Mass Spectrometry
Laboratory for Rho identification, and Andrey Malyutin and
Songye Chen at the Caltech CryoEM facility for their support
during electron-microscopy data collection. We gratefully
acknowledge the Gordon and Betty Moore Foundation and
the Beckman Institute at Caltech for their generous support of
the Molecular Observatory at Caltech. Cryo-electron micro-
scopy was performed in the Beckman Institute Resource
Center for Transmission Electron Microscopy at Caltech. GM/
CA@APS has been funded in whole or in part with Federal
funds from the National Cancer Institute (ACB-12002) and
the National Institute of General Medical Sciences (AGM-
12006). This research used resources of the Advanced Photon
Source, a US Department of Energy (DOE) Office of Science
User Facility operated for the DOE Office of Science by
Argonne National Laboratory under Contract No. DE-AC02-
06CH11357. The EIGER 16M detector was funded by an
NIH–Office of Research Infrastructure Programs High-End
Instrumentation Grant (1S10OD012289-01A1). Use of the
Stanford Synchrotron Radiation Lightsource, SLAC National
Accelerator Laboratory is supported by the US Department
of Energy, Office of Science, Office of Basic Energy Sciences
under Contract No. DE-AC02-76SF00515. The SSRL Struc-
tural Molecular Biology Program is supported by the DOE
Office of Biological and Environmental Research and by the
National Institutes of Health, National Institute of General
Medical Sciences (including P41GM103393).
Funding information
The following funding is acknowledged: Howard Hughes
Medical Institute.
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Figure 4
Single-particle cryo-EM analysis of
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Atm1 samples containing Rho. (
a
)
Size-exclusion chromatograph of the reconstituted
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Atm1 in MSP1D1
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volume is colored gray, while the elution positions of various molecular-
weight standards (in kDa) are marked on the chromatograph. (
b
)2D
classes of Rho illustrating the hexameric arrangement with a diameter of
100 A
̊
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284 A
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