Supporting InformationMiller et al. 10.1073/pnas.0811275106SI Materials and MethodsMacromolecular Synthesis Assay. A hypersensitive strain of Esch-erichia coli (tolC, imp) was used to define the mechanism ofpyridopyrimidine action. Cells were grown overnight in MOPS-defined medium and diluted 1:50 in fresh medium and grown at37 °C for 2.5 h to midlog phase. Test compounds were seriallydiluted in DMSO and transferred to a 96-well cellulose esterfilter plate (Millipore, catalog no. MHABN4550) containing 100�L of MOPS medium in every well. The plate was placed in a37 °C incubator for 10 min to equilibrate, then bacterial culture(100 �L) was added to all wells and incubated for 10 min at 37 °C.Solutions of radiolabeled precursors ([14C]leucine, 0.002 �Ci/mLfinal; [3H]thymidine, 0.04 �Ci/mL final; [3H]uracil, 0.04 �Ci/mLfinal; [14C]sodium acetate, 0.5 �Ci/mL final; or [3H] diamin-opimelate, 0.04 �Ci/mL final) were added to plates that werethen incubated at 37 °C for 15 min. Cold 25% (wt/vol) trichlo-roacetic acid was added to each well [final concentration 6.6%(wt/vol)], and the plate was incubated on ice for 20 min,vacuum-filtered, and washed three times with 200 �L of 5%(wt/vol) trichloroacetic acid. The plate was dried for at least1.5 h, and 30 �L of scintillation mixture was added. Data arecalculated as percentage inhibition relative to cells with noinhibitor and then fit to Eq. s1 to provide an IC50 value.

Details of Spontaneous Pyridopyrimidine-Resistant Mutant-Generat-ing Strains and Their Propagation. Haemophilus influenzae (RD)and Moraxella catarrhalis were propagated on chocolate agar(BBL) in 5% CO2 at 35–37 °C. To avoid mutations associatedwith efflux in H. influenzae, HI 100, a strain with a kanamycincassette inserted into the acrA gene was used and maintained onchocolate medium containing 50 �g of kanamycin per mL. E. colimutants were isolated in JL2, a MC4100 derivative containing aTN10 insertion in outer membrane protein TolC (1) and main-tained on Mueller–Hinton plates containing 10 �g of tetracy-cline per mL.

Determination of Antibacterial Activity: Minimal Inhibitory Concen-trations and Minimal Bactericidal Concentrations. Broth microdilu-tion susceptibility testing was performed by using a BioMek FXrobotic workstation (Beckman–Coulter). Nonfastidious organ-isms were tested in cation-adjusted Mueller–Hinton broth(CAMHB; Becton Dickinson); Streptococci were tested inCAMHB containing 3% lysed horse blood (Remel); Haemophi-lus strains were tested in Haemophilus test medium (PMLMicrobiologicals). All incubations were performed at 35 °C inambient atmosphere.

Generation of Previously Described Protein Reagents. The cloning,expression, and purification of E. coli biotin carboxylase (BC)and Pseudomonas aeruginosa BC have been described elsewhere(2) as has that of E. coli carboxytransferase (CT) (3) and E. colipurine nucleoside phosphorylase (4). The I437T and H438Pmutants of E. coli BC were generated by site-directed mutagen-esis and expressed and purified identically to the wild-typeenzyme.

Production of H. influenzae BC. The acetyl-CoA carboxylase (accC)gene was cloned from H. influenzae genomic DNA (strain RD)and inserted into the pPW2 expression vector (Affinium Phar-maceuticals). This generated a clone with the following sequenceN-terminal to the initiating methionine: MGSSHHHHHHSS-GLVPRGSH. H. influenzae BC was overexpressed in BL21(AI)

cells (Invitrogen) by using modified terrific broth and inductionby arabinose and IPTG at final concentrations of 1 mM and 0.2%(vol/vol), respectively. Induction proceeded for 5 h at 28 °C. Lysisand purification of H. influenzae BC were performed as de-scribed for E. coli BC (2).

Production of Biotinylated E. coli BCCP. The accB gene was ampli-fied from E. coli genomic DNA and cloned into pET202, whichencodes the following sequence N-terminal to the initiatingmethionine: MHHHHHHLVPRGS. This construct was used totransform BL21Star(DE3) E. coli (Invitrogen). Cells were fer-mented in rich medium at 37 °C and until midlog and theninduced with 50 �M IPTG for 24 h at 20 °C. Cell paste (1.09 kg)was resuspended in 3000 mL of 50 mM Tris (pH 8.0), 300 mMNaCl, 1% Triton X-100, 5 mM magnesium chloride, and 110 �Lof benzonase was added. Cells were lysed in a microfluidizer andcentrifuged at 20,000 � g for 90 min at 4 °C. The supernatant wasmixed with 100 mL of washed Ni–nitrilotriacetic acid resin(Qiagen) overnight at 4 °C. The resin was poured into a 5-cm-diameter column and washed with 10 bed volumes of the abovebuffer and then washed with 50 mM Tris (pH 8.0), 500 mM NaCl,20 mM imidazole until the absorbance at 280 nm stabilized. Theprotein was then eluted with 50 mM Tris (pH 8.0), 500 mM NaCl,and 200 mM imidazole. The eluted fractions were pooled anddialyzed overnight at 4 °C into 50 mM Tris (pH 8.0), 150 mMNaCl. In vitro biotinylation was performed in two dialysiscassettes (20-mL capacity), each containing 15 mL of apobiotincarboxyl carrier protein (apo-BCCP; 10 mg/mL). The proteinwas redialyzed against 2 L of 50 mM Tris (pH 8.0), 10 mM MgCl2,5 mM ATP, and 1 mM Tris(2-carboxyethyl) phosphine (TCEP).Into each cassette, 600 �L of 30 �M E. coli BirA (biotin proteinligase, provided by K. Levier, Pfizer, Inc., Ann Arbor, MI) wasadded. To the dialysis solution outside the cassette, 1.5 mL of 20mg/mL Staphylococcus aureus inorganic pyrophosphatase (pro-vided by Z. Xu, Pfizer, Inc., Groton, CT) and 4.4 mL of 228 mMbiotin solution were added.

The apo-BCCP biotinylation reaction was stirred at roomtemperature for 1–2 h and then placed in a cold room wheremixing continued overnight. The next day, 1 mL of sample wasremoved from the dialysis cassettes, and the extent of biotiny-lation was analyzed by electrospray mass spectrometry. Com-plete biotinylation (1 biotin per molecule of BCCP) was ob-served. The protein solution was separated from ATP, BirA, andother contaminants by using a Sephadex 26/60 S200 columnequilibrated in 50 mM Tris (pH 8.0), 150 mM NaCl. Holo-BCCPwas concentrated to 7–10 mg/mL and stored at �80 °C.

Assessment of Inhibitor-Binding Thermodynamics by Isothermal Ti-tration Calorimetry (ITC). Ligand-binding affinities and thermody-namic properties were determined by using a VP-ITC (Micro-cal). Solutions of enzyme (typically 8–12 �M in volumes of 20mL or less) were dialyzed at 4 °C against 2 L of 50 mM Hepes(pH 7.2), 250 mM KCl, and 1 mM TCEP. Ligands to be titratedwere diluted into dialysis buffer from 30 mM DMSO stocks. Allsolutions were thoroughly degassed. Solutions of enzyme (typ-ically 1.8 mL of 8–20 �M) were titrated by successive additionsof 5–10-�L aliquots of ligand (80–200 �M) at 30 °C. Data werecollected and analyzed by using the Origin software package(OriginLab) provided with the instrument (Microcal). Datawere best fit by a model describing an enzyme having a singletype of independent binding site.

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Surface Plasmon Resonance Studies. Kinetic parameters of com-pounds 1 and 2 binding to E. coli BC were determined by usinga Biacore S51 instrument (GE Healthcare). The protein wasimmobilized to CM5 sensor chips at a surface protein level of1,000–4,000 RU by using amine coupling according to standardprotocols. All experiments were performed at 25 °C with a flowrate of 90 �L/min to minimize mass transport effects. Inhibitorswere serially diluted into 50 mM Hepes (pH 7.2) containing 250mM KCl, 1 mM TCEP buffer and 5% DMSO. For eachcompound concentration, triplicate samples were injected at160-s association times and 290-s dissociation times each. Aseries of buffer injections were used as negative control. DMSOcontribution to the measured responses was corrected by usinga standard curve obtained with a DMSO concentration seriesunder the same run. Each dataset was analyzed by using simul-taneous nonlinear regression analysis according to a 1:1 modelwith Biacore S51 BIAevaluation software (Biacore AB). Thereported kon, koff, and Kd values were the average of twoexperiments at different surface protein densities on the sameBiacore chip.

ACCase-Coupled Enzyme Assay. Assays were performed in 384-wellclear bottom plates (Corning; catalog no. 3702), that containedup to 4 �L of inhibitor solvated in DMSO. To each well of theplate 40 �L of a solution (solution 1) consisting of 50 mM Hepes(pH 8.0), 100 mM KCl, 1 mM TCEP, 5 mM MgCl2, 0.1 mg/mLBSA, 0.005% (vol/vol) Tween 20, 30 nM E. coli BC, 50 nM E. coliCT, 50 nM biotinylated E. coli BCCP, and 0.5 unit/mL E. colipurine nucleoside phosphorylase was added. After 5 min ofcoincubation of inhibitors with solution 1, the ACCase reactionwas initiated by addition of 40 �L of solution 2, which consistedof 500 �M citrate (pH 4.2), 150 �M 7-methyl-6-thioguanosine(MESG; Berry and Associates), and 0.005% (vol/vol) Tween 20.Reaction progress was monitored by the increase in absorbanceat 360 nM (Fig. S1).

Inhibitor potency was assessed by duplicate 20-point titrationsof inhibitor from 96 �M to 9.6 nM. Inhibition data were fit to thestandard IC50 equation,

vi

vo�

11 � ��I�/IC50�

n [s1]

where vi is the reaction velocity at a given concentration ofinhibitor [I], vo is the uninhibited velocity, and n is the Hill slope.For inhibitors with IC50 values �50 nM, the data were fit by usingthe Morrison equation (5),

vi

� vo���E�T � �I�T � Kiapp� � ���E�T � �I�T � Ki

app�2 � 4Kiapp �E�T

2�E�T�

[s2]

where vi is the reaction velocity at a given concentration ofinhibitor, vo is the uninhibited velocity, [E]T is the concentrationof enzyme, Ki

app is the apparent inhibition constant, and [I]T isthe inhibitor concentration.

X-Ray Data Collection, Structure Determination, and Refinement.X-ray diffraction data from the crystals were collected in houseor at the Advanced Photon Source facility on beamline 17-IDoperated by the Industrial Macromolecular CrystallographyAssociation (Table S6). The crystals were mounted in thecryoloops and treated with cryoprotection solutions consisting of25% ethylene glycol and the reservoir solution. The crystals weremoved from the crystallization drop to cryosolution for 3–5 sbefore being flash-frozen in liquid nitrogen. Intensity data weremeasured at approximately �180 °C. Autoindexing and process-

ing of the measured intensity data were carried out with theHKL2000 software package (6). The intensity data collectionstatistics are summarized in Table S6. The crystal structures weresolved by molecular replacement by using the coordinates of theAMPPNP-bound structure of E. coli BC as the search model.The rotation/translation searches were carried out with theMOLREP program (7, 8). The molecular replacement solutionwas further optimized by rigid body coordinates and B valueminimization by using REFMAC (7, 9). Calculated (2Fo � Fc)and (Fo � Fc) electron density maps were used for interactivefitting of protein structures into electron density by using theCOOT software program (10). Placement of the ligands intoelectron density maps was carried out with X-LIGAND (11)implemented in QUANTA (Accelrys). Final coordinates werevalidated by using the PROCHECK validation tools (12). Asummary of final refinement parameters and the final Rwork andRfree values are shown in Table S6.

Selection of Bacterial Strains for Time–Kill and Combination Studies.Representative strains of Streptococcus pneumoniae, M. catarrha-lis, and H. influenzae were selected for testing based on theirindicated role in serious upper respiratory tract infections.Additionally, two MRSA strains, SA-1417 (MR 1848) andSA-2017 (EMU-38 CiproR–gyrA S84L, grlA S80Y), were addedbecause MRSA is indicated in Zyvox (linezolid) therapy. The S.pneumoniae isogenic panel includes a clinical strain 7785 (SP-2870) and mutants raised stepwise on clinafloxacin, as well as amouse virulent strain SV-1 (SP-0003). The four isolates of H.influenzae tested include a mouse virulent strain (HI-3543), anacrA� efflux mutant (HI-3927), and two �-lactamase� clinicalisolates (HI-3113 and HI-3542). The two M. catarrhalis strainsinclude an arcA� efflux mutant (BC-4527) and a �-lactamase�

strain (BC-3531). S. pneumoniae and S. aureus cultures weregrown at 35 °C either on tryptic soy agar (TSA) containing 5%sheep blood (BioMerieux Industry) or CAMHB (Becton Dick-inson). For growth of S. pneumoniae, Mueller–Hinton wassupplemented with 5% lysed horse blood (Quad Five). H.influenzae and M. catarrhalis were grown on chocolate agar(BBL; catalog no. 221169). H. influenzae was also cultured inHaemophilus test medium (Remel; catalog no. 112380) and M.catarrhalis in CAMHB, with both species grown at 35 °C and 5%CO2 atmosphere.

Generation of a Nanosuspension of Compound 1 for in Vivo Dosing.Because of solubility limitations, compound 1 was formulatedfor in vivo oral dosing by preparation of a nanosuspension.Compound 1 (90–100 mg/mL) was combined with 0.5-mm glassbeads (Glen Mills) and milling vehicle (2% polyvinylpyrrolidoneK-30, 0.15% sodium lauryl sulfate, 0.005% simethicone) at aratio of 1:10:1.6 in a 4-mL narrow-mouth polypropylene bottle(Nalge). This mixture was attrition milled on a vortex stirrer(model VP708; V&P Scientific) at 2,400 rpm for 18 h by usinga PTFE-encased NdFeB magnet (V&P Scientific). After milling,the nanosuspension was extracted by filtration using a stainlesssteel filter (13-mm diameter; Whatman), diluted with vehicle tothe target concentration and quantitated by UV-visible spec-troscopy.

H. influenzae Peritonitis/Sepsis Model. H. influenzae strain HI-3113(a �-lactamase� clinical isolate) was grown overnight on TSA �5% sheep blood plates. The following day, cells were resus-pended in Haemophilus test medium and mixed with mucin/hemoglobin. Mice (CD-1) were challenged i.p. with 107 cfu/animal. One hour after infection, compound 1 was dosed orallyQD or BID at 200 mg/kg (individual dose amount), and efficacyoutcome was determined from the survival data 7 days afterinfection.

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In Vitro Checkerboard Assays. Test plates were spotted with com-pounds (e.g., 3.2 �L of each compound) by using the Biomek FXor the Multimek 96 MultiChannel Pipettor (Beckman–Coulter).Plates were inoculated with microorganisms (100 �L), dilutedfrom a 0.5 McFarland standard to 2.5 � 105 cfu/mL, andincubated for 16–20 h at 35 °C (ambient atmosphere). Inhibitoryend points were determined by visualization with the aid of a testreading mirror (Dynex Technologies; 220-16). The fractional

inhibitory concentrations (FICs) for various combinations weredetermined by using standard methods, and the FIC index wascalculated for each combination (13). FIC indexes from allindividual combinations in a single plate (minimum drug con-centrations where growth inhibition occurred) were averaged,and the combination interaction was assessed according tostandard nomenclature: additivity (0.5� �FIC �4.0), synergy(�FIC �0.5), or antagonism (�FIC 4.0) (13).

1. Liu JY, Miller PF, Gosink M, Olson ER (1999) The identification of a new family of sugarefflux pumps in Escherichia coli. Mol Microbiol 31:1845–1851.

2. Mochalkin IM, et al. (2008) Structural evidence for substrate-induced synergism andhalf-sites reactivity in biotin carboxylase. Protein Sci 17:1706–1718.

3. Bilder P, et al. (2006) The structure of the carboxyltransferase component of acetyl-CoAcarboxylase reveals a zinc-binding motif unique to the bacterial enzyme. Biochemistry45:1712–1722.

4. Miller JR, et al. (2007) Phosphopantetheine adenylyltransferase from Escherichia coli:Investigation of the kinetic mechanism and role in regulation of coenzyme A biosyn-thesis. J Bacteriol 189:8196–8205.

5. Williams JW, Morrison JF (1979) The kinetics of reversible tight-binding inhibition.Method Enzymol 63:437–467.

6. Otwinowski Z, Minor, W (1997) Processing of X-ray diffraction data collected inoscillation mode. Methods Enzymol, pp 307–326.

7. Collaborative Computational Project, Number 4 (1994) The CCP4 Suite: Programs forProtein Crystallography. Acta Crystallogr D 50:760–763.

8. Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular replace-ment. J Appl Crystallogr 30:1022–1025.

9. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structuresby the maximum-likelihood method. Acta Crystallogr D 53:240–255.

10. Emsley P, Cowtan K (2004) COOT: Model-building tools for molecular graphics. ActaCrystallogr D 60:2126–2132.

11. Oldfield TJ (2001) X-LIGAND: An application for the automated addition of flexibleligands into electron density. Acta Crystallogr D 57:696–705.

12. Laskowski RA, McArthur MW, Moss DS, Thornton JM (1993) PROCHECK: A program tocheck the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291.

13. Pillai SK, Moellering, RC, Eliopoulos, GM (2005) in Antibiotics in Laboratory Medicine,ed Lorian V (Lippincott Williams & Wilkins, Philadelphia), pp 365–440.

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Fig. S1. Schematic representation of the enzyme-coupled ACCase holoenzyme reaction assay.

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Fig. S2. Biophysical assessment of pyridopyrimidine binding to E. coli biotin carboxylase. (A) Binding of 1 to E. coli BC monitored by isothermal titrationcalorimetry (ITC). (Upper) Change in calorimeter power required to maintain constant temperature after each injection. (Lower) Integrated heats of bindingcorrected for heats of dilution. The solid line is the best fit of the data to a single-site binding model. (B) Surface plasmon resonance (Biacore) sensorgrams (coloredtraces) and fitting curves (solid black lines) of compound 1 binding to E. coli BC. The data acquisition and analysis were described in SI Materials and Methods,and the concentrations of 1 (from top to bottom curve) used in the experiments are 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 nM.

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Fig. S3. Schematic 2D representation of protein–ligand interactions in the BC crystal structures drawn by using the MOE program (Chemical Computing Group,CCG). Residues are annotated with their 1-letter amino acid code. Hydrophobic residues are colored with a green interior; polar residues are colored in lightpurple; basic residues are further annotated by a blue interior ring, and acidic residues are further annotated with a red ring. Hydrogen bonding interactionsbetween the receptor and the ligands are drawn with an arrowhead to denote the direction of the hydrogen bond. Hydrogen bonds formed with the residueside chain are shown by the green arrows. Hydrogen bonds formed with the residue backbone are shown by the blue arrows. Solvent-accessible surface areaof the ligands is plotted directly onto the ligand atoms in the form of a blue smudge. (A) Schematic 2D representation of protein interactions with inhibitor 1.(B) Schematic 2D representation of protein interactions with inhibitor 2. (C) Schematic 2D representation of protein interactions with inhibitor 3. (D) Schematic2D representation of protein interactions with ADP (PDB ID code 2j9g).

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Fig. S4. View of the superimposed Connolly binding site surfaces of wild-type BC (magenta) and the I437T mutant enzyme (light blue) in complexes withinhibitor 1. Inhibitor 1 and the residues are shown in sticks with the following atom colors: carbon, green (wild-type EcBC); carbon, yellow (I437T EcBC); nitrogen,blue; oxygen, red; and bromine, purple. The black line denotes where each protein surface is cut away to reveal the expansion of the hydrophobic pocket causedby the resistant I437T mutation.

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Table S1. Frequencies of spontaneous resistance-conferring mutations in the accC gene of various organisms

Organism (genotype) Agar MIC, �g/mL

Resistance frequencies observed at various concentrations of compound 1

2� MIC 4� MIC 8� MIC 16� MIC

H. influenzae 0.2 1 � 10�8 1 � 10�9 1 � 10�9 �2 � 10�9

E. coli (tolC) 0.075 2 � 10�8 9 � 10�9 4 � 10�9 2 � 10�9

M. catarrhalis 1.6 4 � 10�9 4 � 10�9 4 � 10�9 4 � 10�9

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Table S2. Sites of spontaneous resistance-conferring mutations in the accC gene of various organisms

Organism and resistance allele* Selection concentration† Selection compound

H. influenzae (AcrA�)AccC I437T (6) 4, 8, and 16� 1,2AccC I437S (2) 8 and 16� 1,2AccC I437N (8) 16, 32, and 128� 1,2AccC H438P (1) 4� 1

M. catarrhalisAccC I437T (12) 4 and 8� 1

E. coli (TolC�)AccC I437S (3) 2 and 4� 1,2AccC I437N (3) 2 and 10� 1,2AccC I437T (3) 2, 4, and 10� 1,2AccC I157L (3) 2 and 4� 1AccC Y203H (1) 1� 1AccC Y203D (1) 2� 1AccC Y203C (4) 2 and 4� 1AccC Y203S (3) 2 and 4� 1AccC H438P (1) 6� 1AccC H438Q (1) 4� 2AccC H438R (1) 4� 2

*Numbers in parentheses indicate occurrences of each mutation observed. Residue numbering is according to E. coli AccC.†Fold inrease relative to agar MIC observed for parent strain.

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Table S3. Biophysical characterization of pyridopyrimidine binding to BC orthologs

BC ortholog H. influenzae BC compound 2 P. aeruginosa BC compound 1 S. aureus BC compound 2

Kd from ITC, nM �5 6.5 � 1.5 23 � 3�H, kcal/mol �17.46 � 0.01 �15.68 � 0.02 �10.47 � 0.07�G, kcal/mol � (�11.3)* �11.1 �10.6T�S, kcal/mol �6.2* �4.6 0.1

*The parameter T�S is calculated and is a function of �G and �H. In this case, the reported value of �G is an upper limit, and the value of T�S shown is basedon this value of �G.

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Table S4. In vivo efficacy in a murine H. influenzae systemic infection model

Route of administration Total daily dose, mg/kg Dosing frequency Survivors/total

Oral 200, cmpd 1 QD 5/8Oral 200, cmpd 1 BID 3/6Oral 6.25, levofloxacin QD 8/8Oral Vehicle alone QD 0/8

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Table S5. Key active-site residues of biotin carboxylase from different organisms

Organism 116 131 157 159 163 164 165 166 169 201 202 203 204 233 236 276 278 287 288 437 438

E. coli Lys Val Ile Lys Gly Gly Gly Gly Met Glu Lys Tyr Leu Gln His Glu Leu Ile Lys Ile HisH. influenzae K V I K G G G G M E K Y L Q H E L I E I HP. aeruginosa K V I K G G G G M E K F L Q H E L I E I HM. catarrhalis K V I K G G G G M E R F L Q H E L I E I HE. faecalis K V M K G G G G I E K I I Q N E L M E T SS. pneumoniae K V M K G G G G I E R V I Q N E L M E T SS. aureus K V I K G G G G I E K F I Q M E I M E T N

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Table S6. X-ray intensity data statistics and refinement summary

Parameter Compound 1 Compound 2 Compound 3

X-ray diffractionPDB ID code 2V58 2V59 2V5AX-ray source RU200 APS/17BM RUH2RResolution, Å 2.10 (2.18–2.10) 2.40 (2.49–2.40) 2.31 (2.39–2.31)Space group P212121 P212121 P212121

Unit cell a, b, c, Å 84.22, 106.61, 122.34 84.19, 106.20, 123.05 84.32, 106.41, 122.18Complexes per asymmetric unit 2 2 2Observations 326,269 (31,780) 165,893 (9,834) 133,989 (11,504)Unique reflections 63,262 (6,356) 40,822 (3,073) 41,691 (3,967)Multiplicity 5.16 (5.0) 4.06 (3.2) 3.21 (2.9)Rmerge, %* 0.070 (0.423) 0.098 (0.234) 0.070 (0.376)Completeness, % 97.5 (99.5) 93.2 (71.3) 85.3 (83.0)I/� , I 23.98 (4.32) 11.28 (3.38) 15.99 (2.74)

Refinementrmsd from ideal

Bond length, Å 0.006 0.007 0.007Bond angles, ° 0.900 1.023 0.998

No. atoms (chain A/B)Protein (chains A/B) 3449/3447 3425/3428 3445/3447Ligands 20/20 22/22 22Water 547 236 339

Average thermal factors, Å2

Protein (chains A/B) 23.37/26.63 33.47/34.98 26.66/32.58Ligand 26.8/28.6 34.6/52.5 27.67Water 29.89 31.42 27.03

Rwork† 0.1929 0.2084 0.1997

Rfree (5% of data) 0.2187 0.2485 0.2368Ramachandran plot statistics‡

Most favored region 703 (92.5%) 697 (91.2%) 700 (92.1%)Allowed region 55 (7.2%) 63 (8.3%) 56 (7.4%)Generously allowed region 0 (0.0%) 0 (0.0%) 2 (0.3%)Disallowed region 2 (0.3%) 0 (0.0%) 2 (0.3%)

Numbers in parentheses indicate statistics for the high-resolution data bin.*Rmerge �hkl�i I(hkl)i��I(hkl)� /�hkl�I�I(hkl)I�.†Rwork �hkl Fo(hkl)�Fc(hkl) �hkl Fo(hkl) , where Fo and Fc are observed and calculated structure factors, respectively.‡Nonglycine and nonproline residues.

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