Crystal Structure of Plant Photosystem I
Adam Ben-Shem +, Felix Frolow # and Nathan Nelson +*
Department of Biochemistry + and Molecular Microbiology and Biotechnology #,
The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978,
Israel
Supplementary information
Methods
Structure determination
Complete PSI isolated from pea seedlings (Pissum sativum var. Alaska) was
purified in active form and crystallized11. Diffraction data for native and several
heavy atom derivative crystals were collected under cryogenic conditions (100K) at
ESRF beamlines ID14-1 and ID14-4 and processed by the HKL suite41 and CCP4
package42 (Table 1). Most of the 8 heavy atom derivative data sets
(ethylmercurithiosalicylate: three data sets denoted EMTS1-3; Hg-acetate: two data
sets denoted Hg-acetate 1-2; PtCl4: two data sets and uranyl acetate) were not
isomorphous with the native data set. This obstacle has been resolved by a pair-wise
check of isomorphism among all the derivative data sets which revealed that two
putative Pt derivative data sets could serve as the native data set for those Hg
derivatives which were not isomorphous with the native crystal (pseudo-native1 for
EMTS1, pseudo-native2 for EMTS2 and Hg-acetate1-2, and native for EMTS3).
Heavy atom positions for every derivative were determined by isomorphous
difference or anomalous difference fourier maps utilizing external phases, obtained by
molecular replacement method using coordinates of cyanobacterial PSI10 (data not
shown). For phase determination, 5 Hg derivatives, that share the same 8 heavy atom
sites but differ in heavy atom substitution level due to variations in concentrations of
soaking solutions: 0.1 mM – 0.25 mM and soaking times: 1h – 2h, were utilized. In
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addition, the anomalous signal from a uranyl derivative for which no suitable native
or pseudo-native was found and from the 6 intrinsic iron-sulfur clusters (regarded as
single scatterers) were exploited (Table 1). Phases were calculated to 5 Å using
SHARP43 and extended to 4.44 Å using two-fold non-crystallographic symmetry
averaging, solvent flattening and histogram matching as implemented in DM42,44.
Electron density map visualization and model building were performed in O45. The
cyanobacterial reaction center C backbone (without subunits X and M) could be
fitted into the MIRAS (Multiple Isomorphous Replacement Anomalous Signal)
derived electron density map very well. It required no modification in the membrane
section and served as an initial model for the core moiety. Modifications of the core
model in solvent exposed regions were made only if they could be unambiguously
determined in the MIRAS map and where sequence alignment indicated the addition
or deletion of residues. The 15 additional transmembrane helices that could be found
in the MIRAS map were first modeled by C backbone of idealized alpha helical
geometry and then adjusted to better fit the map. Some secondary structure elements
in solvent exposed regions could also be added to the model, which was then
completed by identifying 167 chlorophylls, 3 Fe-S centers and two phyloquinones
(per PSI monomer) in the MIRAS map. Sections of somewhat diffuse electron density
that are situated within the membrane may correspond to additional 8 chlorophylls
that are not included in the model. Rigid body refinement (and TLS refinement of the
core region) were performed in REFMAC 5 (ref. 42). No refinement of individual
residues was undertaken hence the almost lack of difference between R-factor (41%)
and R-free (42%). The structure solution is based solely on electron density maps
calculated using the experimental MIRAS phases after density modification. Figures
were generated by O45 and rendered by Molray46.
Completeness of the C backbone model
In all four LHCI proteins, we were able to trace all transmembrane domains and
virtually all the lumenal exposed regions (except for the loop connecting
transmembrane helices B and C in Lhca1 and very few disordered residues). We
could trace partially the stromal exposed domains, i.e the loop connecting
transmembrane helices A and C and the N-terminal tail. Electron densities
corresponding to non-traced stromal regions are detected at the interface between
LHCI and the core : between Lhca4 and PsaF close to the attachment site of the
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stromal region of helix B to the core (Fig 1a) , between Lhca2 and the stromal region
of PsaJ and between Lhca3 and the stromal region of helix b in PsaA. Deciding
between assignment of these densities to an A-C loop or to an N-terminus is
hampered by ambiguous discontinuities and bifurcations in the map in those areas.
Our model of the reaction center moiety (the core) is missing only very few solvent
exposed regions, most notably the stromal loop connecting the two transmembrane
helices of PsaK (also missing in the cyanobacterial PSI structure). We find an electron
density that probably corresponds to this region but cannot make a definite
assignment. Our conclusions regarding the asymmetric nature of LHCI binding to the
core took into account also unassigned electron densities. Furthermore, since the
transmemrane regions are completely traced it can be asserted that Lhca1 closely
interacts with the core and that only this Lhca protein binds the core within the
membrane.
Assigning the four LHCI monomers and subunits K and G
SDS-PAGE and mass-spectrometer analysis confirm that plant PSI crystals
contain four different LHCI proteins, namely Lhca1-411,40. Lhca1 and Lhca4 are kown
to form a heterodimer that can be reconstituted in-vitro47 while Lhca2 and Lhca3
assemble into either homodimers and heterodimers9,13,14. The two hetero-dimers
present in the structure are therefore assigned to Lhca1-Lhca4 and Lhca2-Lhca3.
Since the association between PsaK and Lhca2-Lhca3 and in particular with Lhca3 is
well documented13,26,48 we assigned the heterodimer near subunit K to Lhca2-Lhca3
(with Lhca3 closer to PsaK) and consequently the other dimer near PsaG to Lhca1Lhca4. The dimerization mode described in the text and in ref.16 dictates that Lhca1
is the monomer tightly bound to PsaG and PsaB with protruding N and C termini
attached to Lhca4. In this arrangement Lhca4 is bound to PsaF, which is in agreement
with ref.49. Antisense inhibition studies of individual LHCI subunits and the recorded
variations in LHCI composition due to changes in environmental conditions also lend
credence to this assignment7,17,26,50.
Due to their sequence and structure similarity the assignment of subunits G
and K to either of the two “poles” of plant PSI could not be based solely on backbone
structure. The proposed arrangement with PsaK retaining its cyanobacterial position
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(and hence PsaG occupies the opposite pole) makes use of the marked differences in
the number of chlorophylls coordinated by these subunits in our structure- PsaG
binds zero or maximally one chlorophyll whereas PsaK binds four. Biochemical data
shows that PsaG binds indeed 0 or 1 chlorophylls and certainly not four15 .
It follows from this proposed arrangement that Lhca3 and Lhca2-Lhca3 are
bound much looser to the core compared to Lhca1-Lhca4. This is supported by
experimental data. Bassi and Simpson51 could prepare PSI depleted of Lhca2-Lhca3
(collectively named then LHC-680) but not of Lhca1-Lhca4 by moderate detergent
treatment of the holo-complex. Ref. 13 and B. Andersen (unpublished results cited
Ref. 13) show that Lhca3 is the LHCI protein most easily lost during preparation of
PSI from barely. Therefore Lhca3 is probably not the LHCI protein whose helix C
forms a helix bundle with one of the core subunits and PsaK, which is known to be
adjacent to Lhca3, is not this core subunit.
Reference
41. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Methods in enzymology 276, 307-326 (1997).
42. Bailey, S. The CCP4 Suite - Programs for Protein Crystallography. Acta Cryst.
D 50, 760-763 (1994).
43. delaFortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter
refinement for multiple isomorphous replacement and multiwavelength
anomalous diffraction methods Methods in enzymology 276, 472-494 (1997).
44. Cowtan, K. & Main, P. Miscellaneous algorithms for density modification. Acta
Cryst. D 54, 487-493 (1998).
45. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved Methods for
Building Protein Models in Electron- Density Maps and the Location of Errors
in These Models. Acta Cryst. A 47, 110-119 (1991).
46. Harris, M. & Jones, T. A. Molray - a web interface between O and the POV-Ray
ray tracer. Acta Crystallographica Section D-Biological Crystallography 57,
1201-1203 (2001).
47. Schmid, V.H., Cammarata, K.V., Bruns, B.U. & Schmidt, G.W. In vitro
reconstitution of the photosystem I light-harvesting complex LHCI-730:
Heterodimerization is required for antenna pigment organization. Proc. Natl.
Acad. Sci. U S A. 94, 7667-7672 (1997).
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48. Jensen, P.E., Gilpin, M., Knoetzel, J. & Scheller, H.V. The PSI-K subunit of
photosystem I is involved in the interaction between light-harvesting complex I
and the photosystem I reaction center core. J. Biol. Chem. 275, 24701-24708
(2000).
49. Haldrup, A., Simpson, D.J. & Scheller, H.V. Down-regulation of the PSI-F
subunit of photosystem I (PSI) in Arabidopsis thaliana. The PSI-F subunit is
essential for photoautotrophic growth and contributes to antenna function. J.
Biol. Chem. 275, 31211-31218 (2000).
50. Zhang H, Goodman HM, Jansson S. Antisense inhibition of the photosystem I
antenna protein Lhca4 in Arabidopsis thaliana. Plant Physiol. 115, 1525-1531
(1997).
51.
Bassi, R. & Simpson, D. Chlorophyll-protein complexes of barley
photosystem I. Eur. J. Biochem. 163, 221-230 (1987).
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Table 1 Statistics for data collection and phase determination
Data collection (Number in parentheses refer to highest resolution shell)
Crystal
Resolution Limit
Rmerge
Completeness
I/(I)
No of Reflections
Redundancy
4.44
4.95
5.20
5.04
5.20
5.24
5.80
4.94
4.98
0.099(0.864)
0.077(0.646)
0.074(0.841)
0.058(0.435)
0.063(0.590)
0.067(0.624)
0.069(0.648)
0.069(0.476)
0.067(0.447)
99.6( 99.5)
99.2( 99.9)
99.0(100.0)
69.4( 67.7)
98.0( 96.8)
98.7(100.0)
98.6(100.0)
97.1( 89.4)
85.1( 85.0)
18.7(2.0)
19.4(2.0)
18.5(2.1)
14.1(2.1)
18.5(2.0)
17.7(2.0)
17.6(2.0)
15.3(2.0)
12.9(2.1)
862552
367121
309528
125210
268246
219386
155485
215201
181347
9.3
5.4
5.3
2.8
4.6
3.7
3.6
3.3
3.1
No. of heavy
atom sites
Total Occupancy
Phasing power
(iso/ano)
Rcullis (centric
reflections)
8
7
8
8
8
2
2.36
1.25
2.80
2.87
2.35
1.11
-/0.81
-/0.73
-/0.76
0.93/0.73
1.47/0.85
1.29/0.85
0.47/0.78
0.64/0.65
-/0.67
0.92
0.80
0.87
0.92
0.97
-
Native
Pseudo-native 1
Pseudo-native 2
EMTS1
EMTS2
Hg Acetate 1
Hg Acetate 2
EMTS3
Uranyl Acetate
Phase determination
Derivative
Native
Pseudo-native 1
Pseudo-native 2
EMTS1
EMTS2
Hg Acetate 1
Hg Acetate 2
EMTS3
Uranyl Acetate
FOM (to 5 Å resolution)
FOM after density modification (phases extended to 4.44 Å resolution)
0.43
0.71
Refinement (Rigid body and TLS)
R-factor
R-free
0.41
0.42
FOM (figure of merit), Phasing Power and Rcullis determined by the programs SHARP or DM
Total occupancy - The sum of refined occupancies of the sites normalized to the occupancy of the iron-sulfur sites
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