unravelling the shape and structural assembly of
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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
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
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.
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
Figure S4. Detai l of the residues at thee re- and si--faces of thee flavin.
5
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
7
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)
8
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.
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