Protein Structure and Folding:
The Structure of CcmP, a Tandem
Bacterial Microcompartment Domain
Protein from the β-Carboxysome, Forms a
Subcompartment Within a
Microcompartment
J. Biol. Chem. 2013, 288:16055-16063.
doi: 10.1074/jbc.M113.456897 originally published online April 9, 2013
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Fei Cai, Markus Sutter, Jeffrey C. Cameron,
Desiree N. Stanley, James N. Kinney and
Cheryl A. Kerfeld
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 22, pp. 16055–16063, May 31, 2013
Published in the U.S.A.
The Structure of CcmP, a Tandem Bacterial
Microcompartment Domain Protein from the
␤-Carboxysome, Forms a Subcompartment Within a
Microcompartment□
S
Received for publication, January 25, 2013, and in revised form, April 4, 2013 Published, JBC Papers in Press, April 9, 2013, DOI 10.1074/jbc.M113.456897
Fei Cai‡§1, Markus Sutter‡2, Jeffrey C. Cameron§3, Desiree N. Stanley§1,4, James N. Kinney‡, and Cheryl A. Kerfeld‡§¶1,5
From the ‡United States Department of Energy-Joint Genome Institute, Walnut Creek, California 94598 and the §Department of
Plant and Microbial Biology and the ¶Synthetic Biology Institute, University of California, Berkeley, California 94720
Background: CcmP is a hypothetical protein conserved among all ␤-cyanobacteria.
Results: CcmP is a ␤-carboxysome component; it forms a bilayered shell protein.
Conclusion: CcmP may facilitate flux of larger metabolites across the carboxysome shell.
Significance: It is the first structure of a ␤-carboxysome tandem BMC domain protein; phylogenetically, it represents a new type
of microcompartment building block.
The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO2 fixation enzymes within a protein
shell. The most abundant carboxysome shell protein contains a
single bacterial microcompartment (BMC) domain. We present
in vivo evidence that a hypothetical protein (dubbed CcmP)
encoded in all ␤-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein,
the first to be structurally characterized from a ␤-carboxysome.
CcmP forms a dimer of tightly stacked trimers, resulting in a
nanocompartment-containing shell protein that may weakly
bind 3-phosphoglycerate, the product of CO2 fixation. The
trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its
orthologs in ␣-cyanobacterial genomes form a distinct clade of
shell proteins. Members of this subgroup are also found in
numerous heterotrophic BMC-associated gene clusters encoding functionally diverse bacterial organelles, suggesting that the
potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense
interest for applications in synthetic biology/metabolic engineering, our results describe a new type of building block with
which to functionalize BMC shells.
□
S
This article contains supplemental Figs. 1– 6, Tables 1– 4, Experimental Procedures, and additional references.
The atomic coordinates and structure factors (codes 4HT5 and 4HT7) have been
deposited in the Protein Data Bank (http://wwpdb.org/).
1
Supported by National Science Foundation Grant MCB0851094.
2
Supported by a postdoctoral fellowship from the Swiss National Science
Foundation.
3
Supported by National Science Foundation Grant EF1105897.
4
Present address: Dept. of Biochemistry, University of California, San Francisco, CA 94158.
5
To whom correspondence should be addressed: United States Department
of Energy-Joint Genome Institute, 2800 Mitchell Dr., Walnut Creek, CA
94598. Tel.: 925-296-5691; Fax: 925-296-5752; E-mail: [email protected].
MAY 31, 2013 • VOLUME 288 • NUMBER 22
Cyanobacteria use solar energy to fix CO2. Like many other
autotrophic organisms, they use 1,5-D-ribulose bisphosphate
carboxylase/oxygenase (Rubisco)6 to catalyze the first rate-limiting step of the Calvin-Benson-Bassham cycle. To survive
under ambient CO2 concentrations with this notoriously inefficient enzyme, cyanobacteria have evolved a CO2-concentrating mechanism to enhance CO2 fixation. The CO2-concentrating mechanism is composed of multiple inorganic carbon
transporters and a proteinaceous organelle, the carboxysome.
Carboxysomes encapsulate Rubisco with carbonic anhydrase
in a protein shell. Based on gene arrangement, the type of
encapsulated Rubisco, and associated proteins, carboxysomes
are divided into two types, the cso- or ␣-carboxysomes and the
ccm- or ␤-carboxysomes. ␣-Carboxysomes are found in the
␣-cyanobacteria (namely marine Synechococcus and Prochlorococcus), and ␤-carboxysomes are found in most other cyanobacteria, which grow in a much wider range of ecological
niches.
The ccm genes encoding ␤-carboxysome proteins are dispersed in several locations in ␤-cyanobacterial genomes (Fig.
1A). The ␣- and ␤-carboxysome share two types of conserved
shell proteins. The major shell constituents are the CsoS1
(␣-carboxysome) and CcmK (␤-carboxysome) proteins; these
contain an ⬃80 amino acid domain called the bacterial microcompartment domain (BMC domain; pfam00936). CsoS1 and
CcmK proteins form hexamers. They self-assemble into layers
that are proposed to constitute the facets (1–5) of the apparently icosahedral carboxysome shell (6 – 8). The shell has been
proposed to be composed of a single layer (2, 4, 5) or a double
layer (9) of hexameric BMC proteins. Based on electron cryotomography studies, in a single layer model, ⬃40 hexamers
would be required per facet to form the complete icosahedral
shell of the average sized ␣-carboxysome of Synechococcus
strain WH8102 (6). This number is estimated to be ⬃30 for the
6
The abbreviations used are: Rubisco, 1,5-D-ribulose-bisphosphate carboxylase/oxygenase; RuBP, 1,5-D-ribulose bisphosphate; BMC, bacterial microcompartment; 3PGA, 3-phosphoglycerate.
JOURNAL OF BIOLOGICAL CHEMISTRY
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␣-carboxysome of Halothiobacillus neapolitanus (10). Both ␣and ␤-types of carboxysome shells contain a minor component,
CsoS4 or CcmL, respectively, that belongs to the EutN domain
family (pfam03319); CsoS4 and CcmL form pentamers proposed to serve as the vertices of the icosahedron (1, 3).
The co-occurrence of pfam00936 and pfam03319 shell protein families is not limited to the genomes of cyanobacteria. A
recent bioinformatic survey revealed that ⬃20% of sequenced
bacterial genomes, from 13 bacterial phyla, have genes coding
for both protein families, suggesting that the potential for forming compartments enclosed by carboxysome-like shells is widespread in the bacterial domain (11). The functions of some of
these have been experimentally determined (12–15), but for
most the only available functional information is derived from
the annotations of the genes clustering with shell protein genes
(3, 11). Collectively, carboxysomes and these (presumably)
architecturally similar organelles are known as BMCs.
Encapsulation of enzymes within a protein shell necessitates
flux of substrates and products across the shell. A key feature of
the current model for carboxysome shell structure and function
are the positively charged pores formed at the 6-fold axis of
symmetry of the hexameric shell proteins. Their diameters
range between 4 and 7 Å, sufficiently large for diffusion of bicarbonate into the carboxysome (2). However, current models do
not explain how the bulkier substrate, 1,5-D-ribulose bisphosphate (RuBP), and the product of CO2 fixation, 3-phosphoglyceric acid (3PGA), cross the shell.
The first structure of a protein containing a fusion of BMC
domains provided a clue (16). This protein, CsoS1D, is encoded
just upstream of the cso operon in most ␣-cyanobacteria.
CsoS1D forms trimers with a relatively large central pore (14 Å
in diameter) of sufficient size for both RuBP and 3PGA to pass.
Two conserved residues (glutamate and arginine) at the 3-fold
axis of symmetry adopt different side-chain conformations,
resulting in either an open or closed pore. Recently, it was demonstrated that CsoS1D is a component of the ␣-carboxysome
(17, 18).
Bioinformatically, an ortholog of csoS1D was identified outside of the main ccm cluster in the genomes of all ␤-cyanobacteria (16, 19). Here, we show that the product of this gene, now
called CcmP, is indeed a constituent of the ␤-carboxysome of
Synechococcus elongatus PCC 7942 (Syn7942) in vivo. We also
report two crystal structures of CcmP at resolutions of 2.5 and
3.3 Å, from two different crystallization conditions; this is the
first structure of a tandem BMC domain protein from a ␤-carboxysome. Evidence from structural, biophysical, and computational analyses suggests CcmP functions to form a gated, discrete nanocompartment within the ␤-carboxysome shell, likely
to conduct larger metabolites. Phylogenetic analysis indicates
that the BMC domains of CcmP and its ␣-carboxysome
ortholog, CsoS1D, belong to a lineage of BMC domain proteins.
Members of this subclass are also found in several distinct bacterial microcompartment gene clusters of heterotrophic organisms; residues for pore gating and formation of a robust dimer
of trimers are conserved in these orthologs suggesting the
capacity to form gated nanocompartments within microcompartment shells is widespread.
16056 JOURNAL OF BIOLOGICAL CHEMISTRY
EXPERIMENTAL PROCEDURES
Strains and Plasmid Construction for Generating Syn7942
Mutants—All DNA fragments were amplified from Syn7942
genomic DNA using primers indicated in supplemental Table
S4 and cloned using a BglBrick strategy (EcoRI-BglII-partBamHI) (20). The cerulean fluorescent protein was first fused to
the C terminus of CcmP before the entire fusion construct
was transferred into a BglBrick-modified neutral site I
(pAM2991䡠SpR) vector containing an isopropyl 1-thio-␤-D-galactopyranoside-inducible Ptrc promoter (21) to generate
Ptrc䡠CcmP-cerulean. To generate PccmK2䡠RbcL-GFP, the ccmK2
promoter (PccmK2) was inserted in front of the RbcL-YFP fusion
to generate a functional transcriptional unit, Ptrc䡠CcmP-cerulean, which was subsequently transferred to a BglBrick-modified neutral site II vector (pAM1573䡠CmR) (22). Transformation of Sy7942 was accomplished as described previously (23).
Cloning, Expression, and Purification—The ccmP gene from
Syn7942 (locus tag Synpcc7942_0520) was obtained by PCR
amplification using Syn7942 genomic DNA as template and the
primers listed in supplemental Table S4. Then it was cloned in
BamHI and AscI sites of expression vector pCOLADuet-1
(Novagen, Madison, WI) via standard digestion and ligation
procedure to generate pCOLA-ccmP. The sequence of the protein coding region was confirmed by DNA sequencing (University of California at Berkeley DNA sequencing facility). Protein
expression and affinity purification of C-terminal His-tagged
CcmP were performed following procedures previously
described (16).
Fluorescence Microscopy—Syn7942 strains harboring Ptrc䡠
CcmP-cerulean and Ptrc䡠RbcL-GFP or Ptrc䡠cerulean alone were
grown on solid BG11 (24) media in an environmental chamber
with an atmosphere of 3% CO2 and a light intensity of ⬃100
microeinsteins/m2 s. 24 h prior to imaging, cells were suspended in BG11 media and spotted onto BG11-agarose (1%)
pads with or without 1 mM isopropyl 1-thio-␤-D-galactopyranoside. Agarose pads were overlaid with a glass coverslip, and
the cells were incubated in the environmental chamber prior to
imaging. Cells were visualized on a plan apochromat (⫻63/1.4
N/A oil) objective using a Zeiss LSM 710 microscope. Image
processing was performed using ImageJ (25).
Crystallization, Data Collection, and Structure Determination—CcmP was crystallized at room temperature using the
hanging drop vapor diffusion method. The protein was in 10
mM Tris-HCl, pH 8.0, at a concentration of 4.4 mg/ml, and 2
mM of 3PGA was added to the protein 12 h before the crystal
tray was set up. Form 1 crystals were obtained by mixing 3 ␮l of
CcmP (with 2 mM 3PGA) with 3 ␮l of reservoir solution of
condition 1 (100 mM sodium acetate, pH 4.6, 1.2 M sodium
formate, and 20% PEG 400). Form 2 crystals were obtained by
mixing 4 ␮l of CcmP (with 2 mM 3PGA) with 2 ␮l of reservoir
solution of condition 2 (17% tacsimate, pH 4.1).
Form 1 or form 2 crystals were rapidly transferred into
mother liquor supplemented with 30% ethylene glycol or 100%
tacsimate, pH 4.1, respectively, mounted in nylon loops, and
frozen by placing them directly into a liquid nitrogen cryostream. Diffraction data were collected at the Advanced Light
Source at Lawrence Berkeley National Laboratory beamline
VOLUME 288 • NUMBER 22 • MAY 31, 2013
Subcompartment within a Microcompartment Shell
FIGURE 1. CcmP is a component of the ␤-carboxysome. A, organization of carboxysomal genes in S. elongatus PCC 7942 (Syn7942). Genes that contain BMC
domain(s) are shown in red. B, wild-type cells harboring CcmP-cerulean and RbcL-GFP were visualized using laser scanning confocal microscopy following
induction of Ptrc䡠CcmP-cerulean with 1 mM isopropyl 1-thio-␤-D-galactopyranoside (IPTG) for 24 h. RbcL-GFP is constitutively expressed using the endogenous
promoter upstream of ccmK2. The chlorophyll-a (chl-a) channel was used as a marker to identify the cell outline. Scale bar, 1 ␮m.
5.0.2. Diffraction data were integrated and scaled with XDS/
XSCALE (26). The structure of CcmP in both crystal forms was
solved by molecular replacement using PHASER (27) implemented in CCP4, using the CsoS1D structure (3F56) as the
search model. Refinement was performed with PHENIX-refine
(28) alternating with model building using 2Fo ⫺ Fc and Fo ⫺ Fc
maps visualized in COOT (29).
Analysis of the Structures—Calculation of least squares root
mean square deviation for comparison of different structures
was performed with Superpose in the CCP4 suite. Protein interfaces were analyzed using PROFUNC and PDBePISA. Structural models were visualized with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.3, Schrödinger, LLC), and
electrostatic surfaces were generated with the APBS2 plugin of
PyMOL (30). Buried surface area was calculated with
AREAIMOL, and shape complementarity was calculated with
Sc (31). Both AREAIMOL and Sc are distributed in the CCP4
suite.
Transmission Electron Microscopy—A 300-mesh Formvar/
carbon-coated copper grid (EMS, Fort Washington, PA) was
floated on a drop of purified CcmP protein for 4 min. The grid
was allowed to air dry and then stained with 2% aqueous uranyl
acetate for 5 min. The negatively stained protein sample was
imaged on a Tecnai 12 microscope at 120 kV, at the Electron
Microscope Laboratory of the University of California,
Berkeley.
Docking of CcmP or CsoS1D into Carboxysome Shell—The
docking of CsoS1D/CcmP into the ␣- or ␤-carboxysome shell
was performed with structural models of shell proteins for both
model organisms, Prochlorococcus marinus strains MED4 and
Syn7942. First, homology models of CsoS1 and CcmK2 were
built using Swiss-Model (32) using CsoS1A from H. neapolitanus (2G13) and CcmK2 from Synechocystis sp. PCC 6803
(2A1B; for single layer) or from Thermosynechococcus elongatus (3SSQ; for double layer) as templates. Subsequently energy
minimization using the Rosetta-Relax protocol was applied to
both the CsoS1D/CcmP hexamer and the CsoS1/CcmK2 layer
MAY 31, 2013 • VOLUME 288 • NUMBER 22
prior to the docking step. Then a Rosetta-Docking protocol (33)
was performed for incorporating CsoS1D/CcmP into CsoS1/
CcmK2 layer. For each docking partner pair, Rosetta-Docking
was performed for more than 5000 independent runs. The best
solution was then used as a starting model in the Rosetta-Docking protocol for another 1000 independent runs, and the results
were plotted to make sure an energy funnel is present (supplemental Fig. S6). A detailed description can be found in the supplemental material.
RESULTS
CcmP Is a Component of the ␤-Carboxysome—A survey of all
available cyanobacterial genome sequences (129 publicly available genomes at Integrated Microbial Genomes) (34) shows
that an ortholog of CsoS1D, annotated as a hypothetical protein, is conserved among all ␤-cyanobacteria. Based on
sequence homology (reciprocal best BLAST hit), its gene product is predicted to be the ␤-carboxysome counterpart of
CsoS1D, recently experimentally confirmed to be a shell protein of the ␣-carboxysome (17). The percentage identity and
similarity between the deduced amino acid sequences of gene
Synpcc7942_0520 from Syn7942 (which contains ␤-carboxysomes) and CsoS1D from Prochlorococcus marinus strain
MED4 CsoS1D is 46 and 69%, respectively. However, the ␤-carboxysome homolog lacks a 50 amino acid N-terminal extension
found in CsoS1D.
Unlike ␣-carboxysomes, ␤-carboxysomes have eluded purification, precluding an inventory of its protein components. To
determine whether the product of Synpcc7942_0520, hereafter
referred to as CcmP (Fig. 1A), is part of the ␤-carboxysome, we
co-expressed a CcmP-cerulean fluorescent protein fusion
(CcmP-cerulean) with Rubisco fused to green fluorescent protein (RbcL-GFP) in Syn7942. Analysis of this strain using fluorescence microscopy shows that CcmP and RbcL are localized
to distinct puncta that are distributed along the long axis of the
cell (Fig. 1B), similar to other fluorescently labeled carboxysome components (23, 35). The values for the Mander’s coeffiJOURNAL OF BIOLOGICAL CHEMISTRY
16057
Subcompartment within a Microcompartment Shell
cients using thresholding are 0.608 and 0.522 for the cerulean
and the GFP channels, respectively; this substantiates the co-localization of CcmP-cerulean and RbcL-GFP. Similar values were
obtained for co-localization of internal carboxysome components
(23). A control strain expressing cerulean fluorescent protein
alone under the same conditions resulted in diffuse signal and no
discrete puncta (supplemental Fig. S1). These results demonstrate
that CcmP is a component of the ␤-carboxysome.
Structure of CcmP—Syn7942 CcmP was expressed with His
tag in and purified from Escherichia coli. It was mixed with
3PGA, a product of Rubisco-catalyzed CO2 fixation, prior to
TABLE 1
Data collection and refinement statistics
Values in parentheses are for highest resolution shell.
CcmP form 1
Space group
Cell dimensions
a, b, c
␣, ␤, ␥
Resolution
CcmP form 2
P21212
P212121
119.35, 137.36, 152.69 Å
90.0, 90.0, 90.0°
39.27 to 2.51 Å
(2.57 to 2.51 Å)
25.6% (112.2%)
7.0(1.6)
97.3% (86.9%)
6.3 (5.2)
1
175.34, 176.20, 200.54 Å
90.0, 90.0, 90.0°
39.41 to 3.30 Å
(3.38 to 3.30 Å)
21.3% (75.9%)
9.71(2.36)
98.6% (96.3%)
4.3 (4.2)
2
25.7%
29.8%
19.5%
22.2%
Ramachandran plot
Most favored region 99.0%
Allowed region
1.0%
Outlier region
0.0%
97.5%
2.5%
0.0%
R merge
I/␴
Completeness
Redundancy
No. of hexamers/
asymmetric unit
Refinement
Rwork
Rfree
crystallization. The protein crystallized in two different conditions with two different orthorhombic space groups (Table 1).
One crystal (form 1) crystallized in space group P21212 with six
monomers in the asymmetric unit and diffracted to 2.5 Å. The
second crystal (form 2) crystallized in space group P212121 with
12 monomers in the asymmetric unit and diffracted to 3.3 Å.
Both structures were solved by molecular replacement using
CsoS1D (3F56) as the search model. The majority of the CcmP
amino acid sequence, residues 3–205 or 206 (of a total of 213
residues), could be modeled into the calculated electron density
for both structures. No residues were observed in the disallowed regions of the Ramachandran plot; 99% and more than
97% of residues are in the most favored region in form 1 and
form 2, respectively (Table 1). The form 1 structure was refined
to final R and Rfree values of 25.7 and 29.8%, respectively. Hexamers (dimers of trimers, see below) of CcmP (Fig. 2A) form
layers in the form 1 crystal lattice, separated by a space that
could accommodate an additional layer. This layer seems to be
occupied by CcmP in different positions; the same phenomenon was previously observed for a different carboxysome shell
protein, CsoS1C (4). This accounts for the relatively high R
values for this resolution. The form 2 structure was refined to
final R and Rfree values of 19.5 and 22.2, respectively; it does not
have a layered crystal packing.
The CcmP monomer is composed of a fusion of two BMC
domains (referred to as N- and C-BMC; Fig. 2B) that share less
than 18% sequence identity to one another but have essentially
identical folds (root mean square difference of 0.77 Å over 345
atoms). Both the N- and C-BMC domains of CcmP are a circular permutation of the typical BMC fold as described previously
FIGURE 2. CcmP contains two BMC domains and forms a hexamer. A, CcmP hexamer shown in ribbons, colored by chain. B, monomer outlined in black was
rotated 90° and rainbow-colored (blue, N terminus; red, C terminus) with 3PGA modeled in and shown as sticks. C, slab view of a CcmP hexamer, with one trimer
(pseudo-hexamer) colored in green and one in cyan. The concave side of each trimer is indicated. D, when a map (␴ of 1.3) was calculated without the presence
of 3PGA molecule in the model, extra electron density was observed in the intra-domain pocket of a CcmP monomer near the conserved histidine residue
(His-18). A 3PGA molecule (shown as sticks) can be credibly fit into this density.
16058 JOURNAL OF BIOLOGICAL CHEMISTRY
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Subcompartment within a Microcompartment Shell
TABLE 2
Comparison of concave-to-concave dimerization interfaces in CcmP, CsoS1D, and CcmK2 structures
Concave-to-concave interactionb
Shape complementarity
Concave-to-concave interface
of two (pseudo)hexamers
Sca
Area included
in calculation
No. of
residues
Å2
CcmP hexamer
CsoS1D (3F56) hexamer
CcmK2 (3SSQ) dodecamer
0.677
0.709
0.762
2583:2599
2096:2083
547:543
Interface
area
No. of
hydrogen bonds
No. of
non-bond contacts
␪ (° angle)c
48
42
0 (12)d
418
310
216
14
15
5
Å2
73:85
70:73
26:26
4171:4120
3642:3642
1363:1363
a
Sc indicates the shape correlation statistic (31) for quantifying the shape complementarity of the protein:protein interface was calculated by using program Sc, which is distributed in the CCP4 suite.
Data were calculated by PROFUNC.
c
The angle ␪ is the rotation required to perfectly align one trimer (pseudohexamer) on top of the other (also see supplemental Fig. S2).
d
Date were described as low barrier hydrogen bonds for 3SSQ dodecamer (9) but were not detected by PROFUNC or PDBePISA.
b
(15, 16, 36 –38). CcmP forms a trimer (or pseudo-hexamer)
with an edge of ⬃36 Å; one side contains a slight depression
centered at the 3-fold symmetry axis, whereas the other side
(concave side) contains a relatively deep depression (Fig. 2C).
One distinct difference between the CcmP trimer and the
CcmK hexamers formed by single BMC domain proteins is that
the size of the aperture at the 3-fold symmetry axis of CcmP is
much larger (⬃13 Å). However, alternate conformations of the
side chains of two absolutely conserved residues near the 3-fold
symmetry axis, Glu-69 and Arg-70, result in CcmP trimers in
which this pore is closed. Interestingly, the open and closed
trimers in the form 1 structure are conformationally distinct,
superimposing with an root mean square difference of 1.2 Å
over 609 C-␣ atoms. A similarly sized gated pore was also
observed in the CsoS1D structure (16). The observation that
the conformations of absolutely conserved residues affect the
size of the pore in multiple structures of both CcmP and
CsoS1D suggest that transport into and out of the carboxysome
through these proteins is controlled by the side-chain conformations of the conserved residues converging at the pore.
Nanocompartment Formed by Dimerization of Two CcmP
Trimers—We observed tightly associated dimers of trimers in
both crystal forms of CcmP (Table 2). The two trimers have a
concave-to-concave orientation (Fig. 2C); residues on the concave side of the trimer are more conserved than on the opposite
side (Fig. 3A). The residues forming the trimer-trimer interface
are primarily found in the permuted region of the primary
structure as follows: helix A and the loop connecting helix A to
␤-strand 2 of both N- and C-BMC domains (Fig. 2B). The two
trimers are not stacked directly upon one another, but one
trimer is rotated ⬃46° (␪CcmP ⫽ 14°; see below and Table 2,
footnote c, and supplemental Fig. S2B) with respect to the other
(Fig. 2A and supplemental Fig. S2B). This results in a staggered
interaction across the interface, so that the N-terminal domain
of a monomer in the upper trimer interacts with the N- and the
C-terminal domain of two different monomers in the lower
trimer (Fig. 2A and supplemental Fig. S2B). The calculated
shape complementarity (Sc) value for the dimer interface of
CcmP is 0.677 (Table 2), which is in the range of Sc value for
antibody/antigen interactions (31). Furthermore, CcmP elutes
as a hexamer in gel filtration (supplemental Fig. S3), and its
thermodynamically stable form in solution is also predicted by
PDBePISA to be a hexamer; the calculated solvation free energy
gain upon dimerization of CcmP trimers is ⫺51.2 kcal/mol, not
including the effect of satisfied hydrogen bonds and salt bridges
MAY 31, 2013 • VOLUME 288 • NUMBER 22
FIGURE 3. Sequence conservation mapped on the CcmP form 1 structure.
Conservation scores were calculated with ConSurf (41). Each amino acid position is assigned a value from 1 (most variable) to 9 (most conserved) based on
their evolutionary rates and then colored according to the grade with yellow
variable and blue conserved. A, external side of pseudohexamer compared
with interface (concave) side; B, side and slab view of hexamer, with 3PGA
molecules shown as spheres and sticks.
across the assembly’s interfaces. Moreover, the concave-toconcave interface is stabilized by 48 hydrogen bonds (Table 2),
of which 36 are contributed by a conserved sequence motif:
NR(X)1–2R(R/K)(G/A)(S/N/Q)(M/L) (residues 176 –183 of
CcmP, supplemental Fig. S5). Comparable values are obtained
for its ␣-carboxysome ortholog CsoS1D (Table 2), and, interestingly, 36 of a total of 42 hydrogen bonds stabilizing CsoS1D
hexamer formation are also contributed by the same sequence
motif. Collectively, these data indicate that the stacked trimer
assembly in both CcmP and CsoS1D is biologically relevant.
Evidence for Metabolite Binding in the Nanocompartment of
CcmP—The dimerization of CcmP trimers across the concave
surface results in the formation of a relatively large internal
compartment, with a volume of ⬃12000 Å3. Access to the inteJOURNAL OF BIOLOGICAL CHEMISTRY
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Subcompartment within a Microcompartment Shell
rior is restricted by the pores at the 3-fold axis of the two trimers; in the form 1 structure, the pore of one trimer is open, and
the second is closed; in the form 2 structure, both pores are
open. The nanocompartment has six identical pockets that may
bind metabolites. The pockets are found within each CcmP
monomer, located at the N- and C-BMC domain interface
(Figs. 2 and 3B), between helix A of the N-BMC and ␤-strand 3
of the C-BMC. Elongated electron density was observed in each
pocket, close to a strongly conserved histidine residue (His-18).
All of the components in the mother liquor as well as cryoprotectant of the form 1 crystallization condition, with the exception of 3PGA, are too small to account for this density. A 3PGA
molecule can be fit with its carboxyl group facing into the
pocket of CcmP in the form 1 structure with an omit map calculated without the presence of the 3PGA molecule in the
model (Fig. 2D). The refined occupancy of the 3PGA was 0.58.
The distance between the carboxyl group of 3PGA and the ⑀-nitrogen atom of the His-18 side chain ranges from 2.4 to 2.8 Å,
which suggests a possible hydrogen bond. We also observed
extra electron density in the same region in the form 2 structure; however, because the crystallization conditions contained
several chemicals with 2– 4 carbon chains, it was not possible to
unambiguously identify the bound molecule. Isothermal titration calorimetry was performed in an attempt to quantify binding affinities between CcmP and 3PGA as well as RuBP. Unfortunately, the measured binding affinities are low and close to
the detection limit of isothermal titration calorimetry (data not
shown); therefore, no conclusive results were obtained. However, from a functional point of view, weak binding and partial
occupancy in the crystal structure is consistent with CcmP’s
proposed function as a conduit for metabolites (addressed
under “Discussion”).
CcmP Incorporation into the Carboxysome Shell—We next
considered how a CcmP hexamer could be fit into models of the
␤-carboxysome shell. There are two possible orientations for
incorporation of a dimer of CcmP trimers into a single layer of
uniformly oriented CcmK2 proteins. First, the CcmP hexamer
can be incorporated into a single layer of CcmK2 by inserting
one trimer in the same orientation as the CcmK2 hexamer (e.g.
incorporated trimer concave side up in a concave-up layer of
hexamers) (Table 3, models ␤1 and ␤3). Alternatively, CcmP
hexamers can be incorporated into a single layer of CcmK2 by
inserting one trimer in the orientation opposite that of the hexamers (e.g. incorporated trimer concave down in a concave-up
layer of hexamers) (Table 3, models ␤2 and ␤4). Taking into
consideration that the open and closed trimers in the form 1
structure are conformationally distinct, modeling was performed with either open or closed trimers for both possible
orientations.
Recently, a concave-to-concave dimerization of CcmK2 hexamers was reported, leading to the proposal that the ␤-carboxysome shell is double layered (9). We examined the fit of a CcmP
hexamer into a layer of CcmK2 dodecamers. Because the
CcmK2 dodecamers did not form layers in the crystal, it was
first necessary to build a layer composed of CcmK2 dodecamers
based on the single layers of hexamers. When the CcmP hexamer is docked into a layer of CcmK2 dodecamers (Table 3,
model ␤5), only one-half of the CcmK2/CcmP interaction is
16060 JOURNAL OF BIOLOGICAL CHEMISTRY
TABLE 3
Comparison of interface between two adjacent building blocks in
models for the ␤- carboxysome shells and in CcmP crystal packing
a
Key symbols for the schematic diagram. CcmP is shown in orange, and CcmK2 is
shown in light blue. In the table, both proteins are shown in side view.
b
Rosetta-Docking was used to find the best solution for each model.
Homology model of CcmK2 hexamer was built based on CcmK2 from Synechocystis sp. PCC 6803 (2A1B). See supplemental material for details.
d
Homology model of CcmK2 dodecamer was built based on CcmK2 from T. elongatus (3SSQ). See supplemental material for details.
e
Adjacent hexamers were observed in CcmP crystal packing (form 1).
c
closely packed; on the opposite side, there are obvious gaps
between the CcmP trimer and the adjacent CcmK hexamer
(supplemental Fig. S4). The inability to incorporate CcmP into
a layer of dodecameric CcmK2 without creating gaps on one
surface can be explained by the difference in the angle ␪, the
deviation from perfect superposition of two (pseudo)hexamers
(supplemental Fig. S2). Whereas ␪CcmP is ⬃14°, ␪CcmK2 is only
5° (Table 2). This 5° angle was sufficient to bring complementarily charged surfaces to stabilize the CcmK2 dodecamer (9),
but the difference between ␪CcmK2 and ␪CcmP precludes tight
interactions between both layers of a CcmK2 dodecamer and a
CcmP hexamer simultaneously. This is also reflected in the resulting buried surface area between the CcmK2 dodecamer and the
CcmP hexamers, which is less than double of the area buried when
the CcmP is fitted into the single layer of CcmK2 (Table 3).
A new model for packing of CcmP hexamers within the carboxysome shell emerges from the form 1 crystal packing, and
CcmP alone forms layers. The surface area buried and shape
complementarity between adjacent CcmP pseudohexamers in
the layer are in the range reported for CcmK2/CcmK2 and
CcmK4/CcmK4 interactions (supplemental Table S1) previously suggested to constitute the facets of the carboxysome
shell (2, 39). Close inspection of the CcmP layer shows that it is
formed by alternating strips of open and closed trimers (Fig.
4A). Self-assembly of CcmP particles is also evident in transmission electron micrographs (Fig. 4B).
BMC Domains of CcmP and CsoS1D Form a Distinct Class—
Although the N-BMC and C-BMC domains of CcmP are structurally similar, they share less than 18% sequence identity.
VOLUME 288 • NUMBER 22 • MAY 31, 2013
Subcompartment within a Microcompartment Shell
FIGURE 4. CcmP packs in layers in the crystal. A, in the form 1 structure,
hexamers of CcmP are packed in layers of alternating orientations of open
(yellow) and closed (blue) trimers. The two side views of the layer show that
strips with same orientations are on the same plane, although the alternately
oriented strips are slightly shifted. B, transmission electron micrograph of
CcmP. Some strips are indicated with red arrows. Scale bar size is 20 nm.
However, each is ⬃40% identical to its ␣-carboxysome counterpart CsoS1D. Noncarboxysomal orthologs to CcmP and
CsoS1D can also be identified in a subset of BMC gene clusters
found in heterotrophic organisms (supplemental Table S2).
Phylogenetic analysis shows that they form a distinct clade of
BMC domains, distal from single BMC domain proteins from
the same organisms (Fig. 5). The N-BMC or C-BMC domains of
CcmP, CsoS1D, and their heterotrophic counterparts group
together, but both N-BMCs and C-BMCs constitute a distinct
family. The pattern, NR(X)1–2R(R/K)(G/A)(S/N/Q)(M/L),
important for forming hydrogen bonding at the trimer-trimer
interface in both CsoS1D and CcmP is also observed in the
primary structure of the heterotrophic orthologs. Interestingly,
in all members of this clade (including noncarboxysomal members), the glutamate and arginine residues for gating the pore
are absolutely conserved (supplemental Fig. S5).
DISCUSSION
Previously, we suggested that a hypothetical protein found in
cyanobacterial genomes was a component of the ␤-carboxysome (16, 19). In this work, we show that it is indeed a part of the
␤-carboxysome (Fig. 1). Structural (Fig. 2) and biophysical
characterization (Table 2; supplemental Fig. S3) suggests that
MAY 31, 2013 • VOLUME 288 • NUMBER 22
CcmP trimers dimerize to form a bilayered building block of the
carboxysome shell. The residues at the trimer-trimer interface
are primarily found in the permuted region of the primary
structure, but permutation does not strictly correlate with
dimerization of tandem BMC-domain trimers. For example, in
the structures of noncarboxysomal tandem BMC domain proteins EutL (3GFH) (38), EtuB (3IO0) (15), and PduB (4FAY)
(40), permutation, but not dimerization, is observed. Notably,
these three proteins lack a conserved amino acid pattern
involved in hydrogen bonding that stabilizes dimerization of
CcmP. In contrast, CsoS1D, which was recently shown to be an
important component of the ␣-carboxysome (17, 18), also contains this motif. CsoS1D, like CcmP, crystallized as a dimer of
trimers with comparable biophysical properties (16) suggesting
that the dimer of trimers of both CcmP and CsoS1D is a building block of both the ␣- and ␤-carboxysome.
Dimerization of the trimers of CcmP and CsoS1D results in
the formation of the nanocompartment, accessible only
through a relatively large pore that is gated by the conformation
of absolutely conserved residues. Co-crystallization of CcmP
with 3PGA resulted in identification of a potential binding
pocket, containing a conserved histidine (supplemental Fig. S5)
for 3PGA within the interior of the nanocompartment (Figs. 2D
and 3B). The low occupancy of the metabolite in the structure
as well as lack of evidence for tight binding (using isothermal
titration calorimetry) suggest the metabolite is only transiently
retained, which is consistent with the function of the pore and
nanocompartment as a passageway into the carboxysome. A
similarly sized depression containing a conserved histidine residue (His-172) can also be identified in CsoS1D (supplemental
Fig. S5). However, it is not located at the interface of N-BMC
and C-BMC domains, as in CcmP, but at the interface of two
adjacent monomers.
Unlike the pores of the single BMC domain hexameric shell
proteins, the central pore of CcmP or CsoS1D is large enough
for RuBP and 3PGA to readily enter. However, if such large
pores were constitutively open, some of the advantage of the
diffusive resistance to loss of substrate provided by the carboxysome shell would be negated. Perhaps the presence of a nanocompartment gated on both sides functions analogously to an
airlock; the compartment served as a foyer where the metabolite enters through the open pore facing the cytosol and is temporarily retained in the compartment. Subsequently, the pore
open to the cytosol closes, and the other pore facing the carboxysome interior opens. Interestingly, in one of the CcmP
structures reported here, as well as in structures of CsoS1D
(16), each hexamer contained one trimer with a pore in the
open conformation, whereas the pore of the second trimer was
closed.
In this model, CcmP is a specialized shell protein for the
passage of larger metabolites. The bulk of the carboxysome
shell is thought to be composed of single BMC domain protein
hexamers (2, 5). The CcmP dimer of trimers can be fit into
single layer models of the carboxysome shell (Table 3); however, one trimer of the pair is left out of the plane of the layer.
Comparable values for fitting CsoS1D into a single layer of
CsoS1 in the ␣-carboxysome shell are obtained (supplemental
Table S3).
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Subcompartment within a Microcompartment Shell
FIGURE 5. Phylogram of BMC domains. BMC domain containing proteins from ␣-carboxysomes (Cso, labels in black), ␤-carboxysomes (Ccm, labels in dark
gray), and heterotrophic bacteria, where orthologs to CcmP are found (other, labels in light gray), are shown in an un-rooted distance tree. Two BMC domains
(N-BMC and C-BMC) of CcmP, CsoS1D, and their heterotrophic orthologs are constructed separately. Bootstrap values were obtained from 100 replicates.
Numbers above the branches indicate bootstrap values, and only branches receiving bootstrap values greater than 49 are indicated.
Recently, it has been suggested that the entire carboxysome
shell is composed of a double layer of hexameric shell proteins
(9). The major building block of these layers is proposed to be a
CcmK2 dodecamer. A comparison of the CcmK2 dodecamer
with the CcmP hexamer shows that they differ in the amount of
surface area buried and the number of hydrogen bonds in the
interface (Table 2), suggesting that the dimerization of hexamers of CcmK2 is less robust than the dimerization of trimers in
CcmP. A key difference between these two building blocks,
with implications for shell assembly, is how symmetrically the
trimers of CcmP or hexamers of CcmK2 are appressed. Because
of the difference in ␪ (Table 2 and supplemental Fig. S2), a
CcmP hexamer fits closely with only half of an adjacent CcmK2
dodecamer. In contrast, crystal packing (Fig. 4A) and transmission electron micrography (Fig. 4B) suggest that CcmP hexamers are capable of higher level assembly. In the observed layers
and strips, the stacked trimers of CcmP create isolated nanocompartments (Fig. 3B). In contrast, because of the relatively
loose association in the hexamer䡠hexamer interface in dodecameric CcmK2, the space formed between the two layers is continuous (Fig. 3 in Ref. 9).
Based on phylogenetic analyses, the primary structures of
the N- or C-BMC domains of CcmP and CsoS1D form distinct clades of BMC shell proteins distal to their single BMC
domain counterparts (e.g. CsoS1s and CcmKs; Fig. 5).
Accordingly, the emergence of CcmP or CsoS1D is likely to
have been an ancient event, before the separation of the ␣and ␤-carboxysomes, and it supports our conclusion based
on their similar structural properties (Table 2) that they are
functionally equivalent.
Phylogenetic analysis also suggests the ability to form gated
nanocompartment shell building blocks may extend to other
bacterial microcompartments besides carboxysomes, where
16062 JOURNAL OF BIOLOGICAL CHEMISTRY
they may likewise be necessary for conducting relatively large
metabolites across microcompartment shells without compromising the diffusive resistance afforded by a shell. Several subsets of the heterotrophic microcompartment groups (supplemental Table S2; and see Table 1 in Kerfeld et al. (11) and
supplemental Table S3 in Kinney et al. (23)) also contain genes
encoding tandem BMC domain proteins that cluster with
CcmP and CsoS1D (Fig. 5). Many of the residues involved in the
dimerization of trimers are conserved, and strikingly, the glutamate and arginine residues involved in gating the pore are
also absolutely conserved (supplemental Fig. S5). This suggests
that these BMCs of unknown function have shell proteins that
also form nanocompartments with a large, gated central pore.
Given the growing interest in BMCs for synthetic biology, an
understanding of the structural and biophysical properties of
the distinct shell protein building block represented by CcmP
should be useful in informing the design of subcellular enzymatic reactors based on BMC architectures.
Acknowledgments—We thank the staff at the Advanced Light Source,
Lawrence Berkeley National Laboratory, which is supported by the
Director, Office of Science, Office of Basic Energy Sciences, of the
United States Department of Energy under Contract No. DE-AC0205CH11231. We thank Annette Salmeen for help in synchrotron data
collection and Kent McDonald and Reena Zalpuri at the Electron
Microscope Laboratory of the University of California, Berkeley, for
help with electron microscopy. We thank Seth D. Axen and Kirsten
Fagnan for help with Rosetta-Docking and running modeling jobs on
the supercomputing cluster Genepool at The National Energy
Research Scientific Computing Center. We also thank Seth D. Axen,
Jonathan Lassila, and Beth Wurzburg for critical reading of the
manuscript.
VOLUME 288 • NUMBER 22 • MAY 31, 2013
Subcompartment within a Microcompartment Shell
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