4548 Nucleic Acids Research, 2009, Vol. 37, No. 14 Table 2. Diﬀraction data and reﬁnement statistics for full-length CrgA Crystal form A Crystal form B ID14–4 P212121 Q315r ADSC CCD Native FL-CrgA X-ray source Space group Detector Unit-cell parameters a (A˚ ) b (A˚ ) c (A˚ ) b (8) Wavelength (A˚ ) Resolution range (A˚ ) Last resolution shell (A˚ ) Unique reﬂections Completeness (%) Redundancy Average I/ (I) Rmergeb Reﬁnement Resolution (A˚ ) No of reﬂections Rwork/Rfree Number of atoms Rmsd bonds (A˚ ) RMSD angles (8) B Wilson (A˚ 2) Mean B factor Ramachandran plot Most favoured (%) Allowed (%) Disallowed (%) 104.64 119.09 250.20 0.9765 30.0–3.20 3.31–3.20 43 934 (4398) 83.8a (85.3) 3.7 (2.7) 17.8 (1.66) 0.061 (0.512) 29.9–3.2 40 596 21.8/28.6 18304 0.006 0.9 96.5 83.5 95.6 4.2 0.3 Values in parentheses are for data in the highest resolution shell. abRRmeseorgleu=tioPnshell hkl Pi 3.6–3.79 | Ii(hkl)–hI(hkl) A˚ is 27.2% |/Phkl complete Pi Ii(hkl). due to ice rings. buﬀer, the samples were loaded on to a non-denaturing polyacrylamide 4–20% TBE gel and electrophoresed in 0.5 TBE running buﬀer at 120 V for 1.5 h. The gel was stained for DNA with SYBR Green EMSA gel stain (Invitrogen). The CrgA:DNA complex was formed by elution of CrgA in the presence of excess DNA of the 63-bp CrgA sequence footprint, from a 16/60 Hiload superdex 200 column (GE healthcare). Mass spectrometry Nano-electrospray MS and IM-MS of CrgA, DNA and CrgA:DNA complexes were acquired using either a Qstar XL (MDS Sciex, Applied Biosystems, Concord, ON, Canada) or a Synapt HDMS (Waters, Manchester, UK) under conditions optimized for the transmission of non- covalent protein complex interactions using previously described protocols (38,39). Apo and holo protein was buﬀer exchanged using 5 kDa molecular weight cut-oﬀ Vivaspin columns (Vivascience) into 200 mM ammonium acetate, pH 6.8, in order to mimic the ionic strength of the original buﬀer. DNA was exchanged into 200 mM ammo- nium acetate using micro bio-spin 6 columns (BioRad). Typical instrument parameters, in positive ion mode, on the Qstar XL for apo and holo CrgA were: ion spray voltage 1.2–1.65 kV, de-clustering potential 200–250 V, focusing potential 200 V, de-clustering potential 2 15V, P21 123.17 204.99 124.80 93.10 0.9765 30.0– 3.80 3.94–3.80 60 401 (5853) 99.6 (97.2) 3.7 (3.3) 10.4 (4.10) 0.138 (0.365) quadrupole voltage (Q0) 100–200 V, collision gas (CAD) 12, ion release delay 6 and ion release width 5. Experiments were acquired at instrument base pressure of 6.5 mbar. Typical instrument parameters, in positive ion mode, on the Qstar XL for apo DNA were: ion spray voltage 1.2 kV; de-clustering potential 100 V; focusing potential 150 V; de-clustering potential 2 15 V; quadrupole voltage (Q0) 50 V; collision gas (CAD) 3; ion release delay 6 and ion release width 5. Typical instrument parameters for IM-MS, using a Synapt HDMS, in positive mode, were: capillary voltage 1.7 kV; sample cone 40 V; extractor cone 0 V; trap collision energy 10 V; transfer collision energy 10 V; bias voltage 20V; backing pressure 4.3 mbar; trap pressure 5.3 10 2 mbar; T-wave drift cell pressure 5.1 10 1 mbar and ToF pressure 1.8 10–6 mbar. For activation experiments voltages were increased gradually to sample cone 200 V (in 20 V steps), extraction cone 10 V (in 2 V steps), trap collision energy 200 V (in 20 V steps) and bias voltage 180 V (in 20 V steps). Data were acquired and processed using Analyst QS (Applied Biosystems, Concord, ON, Canada) and Masslynx software 4.1 with driftscope (Waters, Milford MA, USA). All spectra are shown with minimal smooth- ing. Spectra were calibrated externally using a 100 mg ml–1 solution of caesium iodide solution. Analytical ultracentrifugation Sedimentation velocity experiments were performed in a Beckman Optima XL-I analytical ultracentrifuge, as pre- viously described (40). The experiment was performed at 40 000 rpm in an An60Ti rotor at 208C. The partial speciﬁc volume used was 0.73 ml g–1. The buﬀer density was 1 g ml–1 and the viscosity 0.01002 poise. Protein samples were prepared in 20 mM Tris pH 7.5, 200 mM NaCl. The sed- imentation proﬁles of samples of CrgA (1.9 mM) alone and in the presence of an equimolar amount of the 63 bp DNA probe were observed over time using interference optics, since the signal in the absorbance trace was dominated by that from the DNA. Analysis by the c(s,f/f0) method used the software SEDFIT (41,42). The c(s) is a method for model-independent analysis of velocity boundary proﬁles whereby an apparent distribution of sedimentation coeﬃ- cients, apparent as a Gaussian distribution, is shown by each sedimenting species or set of species (a set of species if interconverting rapidly on the timescale of the experi- ment). The Gaussian distribution derives from the eﬀects of diﬀusion on the apparent sedimentation coeﬃcient, as aﬀected by the changing concentration of the sedimenting species in the boundary. c(s,f/f0) Distributions are two dimensional plots of the relationship between sedimenta- tion coeﬃcient and frictional ratio (f/f0), which can be inferred from the degree of broadening in the boundary. The proportions of species observed with diﬀerent S values were calculated by integrating the areas under the peaks of two dimensional plots. The function c(s,f/f0) can then be used to derive functions such as c(s,M), dependence of sedimentation coeﬃcient and weight, and c(s,Rs), dependence of