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.