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Acta Cryst. (2015). D71, doi:10.1107/S1399004715018520 Supporting information
Volume 71 (2015)
Supporting information for article:
Unravelling the shape and structural assembly of photosynthetic GAPDH–CP12–PRK complex from Arabidopsis thaliana by small-angle X-ray scattering analysis
Alessandra Del Giudice, Nicolae Viorel Pavel, Luciano Galantini, Giuseppe Falini, Paolo Trost, Simona Fermani and Francesca Sparla
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Figure FMNATCaFADactive scartoon
S1. (A) CT module coS dimer of
sites of diffen and the re
Cartoon repoloured in g
trimers shoerent protomst as surfac
presentatiogreen and thowing the hmers withinces.
n of the Che RFK mohead-tail ar the trimer.
aFADS module in oran
rrangement One of the
onomer (PDnge. (B) Reand the int
e protomers
DB 2X0K), epresentatioteractions bs is represe
2
with the on of the between ented as
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Figure and moresidueat the tFlapI aRFK froshown coloureyellow).
S2. (A) Sonfunctiona
es of prokartop. (B) Carnd L4c-Flaom S. pombin red. (D)
ed with carb. The ADP m
Sequence aal RFKs. Cryotic RFKsrtoon represpII are highbe in comp
Detail of tbons in bluemolecule bo
alignment oonserved m are coloursentation ohlighted in lex with ADthe conforme) and the Round to S. p
of the RFK motifs are red in red. Sof the free R
red. (C) CDP. The sammation of thRFK from Spombe RFK
modules ofcoloured inSecondary RFK module
Cartoon reprme two reghe PTAN eS. pombe (CK is shown i
f selected bn purple anstructure ele of CaFADresentation
gions highlignvironmentCPK colourn transpare
bifunctionalnd yellow. lements are
DS. The looof monofu
ghted in B t in CaFADred with caent yellow C
3
FADSs Specific
e shown ops L1c-unctional are also
DS (CPK rbons in
CPK.
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Figure CaFADMg2+ arHsRFK 1P4M) also shconform(CPK cwith caand Hsas blue
S3. (A) S (blue; PDre shown in
in compleand co-crys
hown in redmation of thcoloured witrbons in ligsRFK are sh and light b
Cartoon reDB 5A89). Ln CPK with ex with FMstallyzation d. FMN ande PTAN enth carbons ght brown). hown in lighrown/ green
epresentatioL1c-FlapI ancarbons in N and ADP(green; PD
d ADP-Mg2
nvironment in blue) anThe FMN a
ht and dark n spheres fo
on of the tnd L4c-Flapblue. (B) C
P-Mg2+ as DB 1Q9S). T
+ in CPK win the terna
nd in the teand ADP-Mgrey CPK, or CaRFK m
ternary compII are highlCartoon repobtained b
The same twwith carbonary complexrnary comp
Mg2+ molecurespectivel
module and
mplex of thighted in re
presentationby soaking wo regions hs in orange
x of the CaFplex of HsRules bound y. Mg2+ atom HsRFK, re
he RFK moed. FMN ann of monofu
(light browhighlighted e (C) DetaFADS RFK
RFK (CPK cto CaRFK
ms are represpectively.
4
odule of nd ADP-unctional wn; PDB
in A are il of the module
coloured module
resented
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Figure S4. Detai l of the residues at thee re- and si--faces of thee flavin.
5
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Figure complexelectronCa2+ co
x. (B) Detan density moordination i
S5. (A) 2FoFc elec tailed view
map around in the binary
tron densityof the MgADP and Cy complex.
y map arou2+ coordinaCa2+ in the
und FMN, Aation in thebinary com
ADP and Me ternary c
mplex. (D) D
Mg2+ in thecomplex. (CDetailed view
6
ternary C) 2FoFc
w of the
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Figure S6. (A) Zn2+ binding in the ternary complex. (B) Coordination of the Zn2+ ion. (C) 2FoFc electron density map around Zn2+ ion. (D) Anomalous map around the Zn2+ion.
Figure S7. Steady-state rates for the RFK activity of ∆(1-182)CaFADS as a function of (A) the RF concentration at saturating ATP concentrations and (B) as a function of the ATP concentration at RF concentration giving maximal activity. All the experiments were assayed in 20 mM PIPES, 0.8 mM MgCl2, pH 7.0 at 25 ºC.
A B
C D
H2O
H186 H325
D321
Zn2+
0 100 200 3000
5
10
15
20
25
k obs (
min
-1)
[ATP] (M)
0 5 10 15 200
5
10
15
20
25
30
35
40
k obs (m
in-1)
[RF] (M)
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Supporting Table
Table S1 Crystal structures of proteins involved in FMN and FAD biosynthesis.
Organism Protein PDB codeEukaryotesHomo Sapiens RFK:ADP-Mg2+
RFK:RF:ADP-Mg2+ RFK:FMN:ADP-Mg2+ (1) RFK:FMN:ADP-Mg2+ (1)
1NB0 1NB9 1P4M 1Q9S
Schizosaccharomyces pombe
RFK RFK:ADP RFK:ADP:FMN RFK:ADP-Zn2+
1N05 1N06 1N07 1N08
Tripanosoma brucei RFK 3BNW Candida glabrata FMNATeukaryote
FMNATeukaryote:ATP FMNATeukaryote FMN:AMP-CCP FMNATeukaryote FAD:PPi
3FWK 3G59 3G5A 3G6K
Saccharomyces cerevisiae FMNATeukaryote:FAD 2WSI Archaeas Methanocaldococcus jannaschii
RFKarchaea RFKarchaea P RFKarchaea: P:CDP RFKarchaea: CDP RFKarchaea:CDP:FMN
2P3M (RMN) 2VBS 2VBT 2VBU 2VBV
Thermoplasma acidophilum RFKarchaea 3CTA
Prokaryotes Corynnebacterium ammoniagenes FADS:PPi 2X0KThermotoga maritima FADS
FADS, crystal form II FADS:lumichrome FADS:ADP FADS:ADP:AMP:FMN FADS:RF
1MRZ 2I1L 1S4M 1T6X 1T6Y 1T6Z
Streptococcus pneumoniae FADS(2) 3OP1
(1) Both RFKs and FADS co-purify with flavin cofactors, which are removed during purification process. The structure of the ternary complex was solved with the protein co-purified with both products (1Q9S) and with crystals of apo-protein soaked with ligands (1P4M), resulting in two different structures.
(2) Crystal structure of the FADS from S. pneumoniae is annotated in the PDB as Macrolide-efflux protein, although it is a bifunctional FAD synthetase. It contains many small molecules located in the active sites, suggesting a residual occupation with flavin and/or adenine nucleotides that might co-purify with the protein.