De novo modeling of the F(420)-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy.

Cultivation Methanothermobacter marburgensis (DSM 2133) was obtained from the Deutsche Sammlung von Mikroorganismen (DSMZ, Braunschweig, Germany). The archaeon was grown anaerobically at 65°C on 80% H 2 /20% CO 2 /0.1% H 2 S in a 12-L fermenter containing 10 L complete mineral salt medium ( Schönheit et al., 1980 ). Cells were harvested by the use of a continuous-flow centrifuge under anoxic conditions at the late exponential phase and stored at −80°C.

Purification of the Frh complex from M. marburgensis

Purification was performed under strictly anaerobic conditions at 18°C in an anaerobic camper (Coy Laboratory Products, Grass Lake, MI). All buffers used contained 2 mM DTT and 25 µM FAD. Cell extracts were routinely prepared from 20 g (wet mass) of M. marburgensis cells. The cells were suspended in 35 ml 50 mM Tris/HCl pH 7.6 (buffer A), and passed four times through a French pressure cell at 125 MPa. Cell debris were removed by centrifugation at 15,000×g for 30 min. The supernatant, designated cell extract and containing ∼1000 mg protein, was adjusted to 800 mM ammonium sulfate in buffer A and stirred for 20 min. The cell extract was applied to a Phenyl Sepharose 6 Fast Flow column (6 × 10 cm) equilibrated with 800 mM (NH 4 ) 2 SO 4 in buffer A. Protein was eluted by a (NH 4 ) 2 SO 4 step gradient in buffer A: 800 mM (NH 4 ) 2 SO 4 for 250 ml, 200 mM (NH 4 ) 2 SO 4 for 250 ml, and 0 mM (NH 4 ) 2 SO 4 for 50 ml (flow rate: 8 ml/min). The Frh activity was eluted in the 0 mM (NH 4 ) 2 SO 4 fractions. The protein solution was concentrated with ultrafiltration by Amicon filters (100-kDa cutoff) to 4–5 ml, which were then applied to a Sephacryl S-400 HR column (2.6 × 60 cm) equilibrated with buffer B (buffer A + 150 mM NaCl). The Frh activity was eluted after washing the column with 180 ml buffer B (flow rate: 1 ml/min). The pooled fractions were concentrated with ultrafiltration by Amicon filters (100-kDa cutoff) to 20 mg/ml. CHAPS was added to the concentrate (48 mM final concentration), and the solution was incubated for 12 hr at room temperature with slow stirring. The protein solution was washed 5 times on Amicon filters (100-kDa cutoff) with buffer A with 4 mM CHAPS (buffer A2) and applied to a MonoQ column (1 × 8 cm) equilibrated with buffer A2. Protein was eluted by a NaCl linear gradient in buffer A2: 0–400 mM NaCl in 25 ml and then 400–600 mM NaCl in 20 ml (flow rate: 0.8 ml/min). In the linear gradient of 400–600 mM NaCl, the active Frh was eluted from the column at a concentration of 540 mM NaCl. The protein solution was concentrated on Amicon filters (100-kDa cutoff) to 2 ml and further purified on a Sephacryl S-400 HR column in order to remove remaining smaller particles and bigger aggregates. F 420 was isolated from M. marburgensis by established methods ( Shima and Thauer, 2001 ). Cryo-electron microscopy 3 µl of a 0.7 mg/ml Frh sample was applied to freshly glow discharged Quantifoil R1/4 grids (Quantifoil Micro Tools, Jena, Germany) that had been pretreated for 15 s in chloroform. The |grids were blotted in an FEI Vitrobot using a 2.5 s blotting time at 70% humidity and 10°C and plunge-frozen in liquid ethane. For collection of data of Frh in the presence of substrate, 0.5, 1.0, or 10 mM F 420 was added to the sample under oxygen-free conditions just prior to grid preparation. Images were collected at liquid nitrogen temperature on an FEI Tecnai Polara operated at 200 kV. Before images were recorded, the microscope was carefully aligned in an iterative process to correct for objective astigmatism and beam tilt by coma-free alignment ( Glaeser et al., 2011 ). The corrections were carried out at a dose of 15 e − /Å 2 and at half the defocus value used for collecting the images, and were repeated for each grid square from which images were collected. The alignments were done with a Gatan 4k × 4k CCD using unbinned images. Images were recorded on Kodak SO-163 film at a magnification of 59,000× with a dose of 10–15 e − /Å 2 at a defocus of 1.5–2.8 µm. The film was developed for 12 min in full-strength Kodak D-19 developer and fixed for 8 min in Kodak Rapid Fix. Five hundred and six negatives were collected of substrate-free Frh and 512 of Frh with F 420 . Films that showed obvious flaws (too thin ice with particles just around the edge of the Quantifoil hole, too high particle concentration, broken ice, obvious drift) were discarded. Four hundred and two negatives of substrate-free Frh were scanned and 277 of F 420 with Frh. The latter preparation had a higher protein concentration, resulting in many films with too many overlapping particles. This was more than compensated by the higher number of particles per film.

Image processing

Films were digitized on a Zeiss Photoscan scanner with a pixel size of 7 µm, corresponding to 1.14 Å on the specimen as calibrated with fatty acid synthase ( Gipson et al., 2010 ). Particle selection was done semiautomatically with the Boxer module from EMAN ( Ludtke et al., 1999 ) and data processing with EMAN2 ( Tang et al., 2007 ). The contrast transfer function (CTF) of the selected particles from each film was determined with EMAN2 and images that showed visible Thon rings in the power spectrum to high resolution and no indication of drift or astigmatism were selected for further processing and their CTF was corrected by phase flipping. A tetrahedral starting model was created by the EMAN2 program e2initialmodel from a number of class averages. This model was iteratively refined using the main EMAN2 program e2refine, which determines the 3D orientation of each particle by comparison to a set of projections of the current 3D reference map. Particles in the same orientation are aligned and averaged, and a new 3D map is constructed from the averages that are then reprojected to create references for the next refinement cycle ( Ludtke et al., 1999 ; Tang et al., 2007 ). Tetrahedral ( T ) symmetry was applied throughout. In the initial refinement steps, the data were binned to a pixel size of 2.28 Å. After the resolution reached ∼10 Å, the unbinned data were used. At every refinement step, the 30% worst members of each class were discarded. Because of the high symmetry and globular shape of the particle, all class averages had similar quality (see Figure 1B ) and were used for each reconstruction. The final data set of substrate-free Frh contained 84,000 particles from 101 negatives of the 402 scanned negatives. Between iterations, the projection angle was varied. In the last refinement step an angle of 0.9° between consecutive reference reprojections was used, yielding 2134 reference images. The F 420 data were refined with a high-resolution map of substrate-free Frh as a starting reference map. Ninety seven thousand particles were selected from 80 negatives. Smaller datasets of Frh with 0.5, 1.0, and 10 mM F 420 were used initially, but no significant differences were seen and the datasets were merged. The resolution of the maps was estimated using the command eotest in EMAN2, which calculates the Fourier shell correlation (FSC) between reconstructions made by half-data sets from the odd and even numbered particles. This indicated 3.9 and 4.0 Å for the maps without and with F 420 , respectively, using the 0.5 FSC criterion ( Figure 10A ). The resolution estimate can be inflated by the overfitting of noise at high resolution ( Grigorieff, 2000 ; Scheres and Chen, 2012 ). To test for this effect, we used the procedure e2refine-evenodd in EMAN2, where the phase of the starting model are randomized from a cutoff resolution lower than the expected resolution of the map, and then two half-data sets are refined to convergence against this model. High-resolution noise will be uncorrelated between the two maps. Subsequently, the FSC between the two resulting maps is calculated, and the resolution from these two independent half-data sets determined at 0.143 FSC ( Rosenthal and Henderson, 2003 ). This procedure yielded a resolution of 5.5 Å ( Figure 10B ). B-factors of the reconstructions were estimated with the program embfactor ( Rosenthal and Henderson, 2003 ; Fernández et al., 2008 ) in the resolution range 10–4.5 Å as 54 Å 2 for the substrate-free map and 60 Å 2 for the map with F 420 (see Figure 10D ). Model building Maps were visualized in Chimera ( Pettersen et al., 2004 ). Homologous protein structures were fitted manually in the map followed by a rigid body fit (pdb 2wpn, Marques et al., 2010 , for FrhA and the C-terminal domain of FrhG; pdb 1dur for the ferredoxin domain of FrhG). Models were then mutated to the correct amino acid and manually rebuilt in Coot ( Emsley and Cowtan, 2004 ). Regions for which no homolog was available, including all of FrhB, was built ab initio in Coot, based on secondary structure predictions done using the PSIPRED server ( Bryson et al., 2005 ). The conserved cysteines serving as ligands for the Fe-S clusters and the visibility of the Fe-S clusters in the map gave clear initial hints to the identity of secondary structure elements. Helices were built using the command Place helix . Loops and β-sheets were built by first placing C α atoms using the Baton build module. Full-atom models were built based on the visibility of side chains, which led in all cases to an unambiguous assignment. The models were refined using the Regularize Zone module. Where possible, side chains were fitted to available density (overall about 55% of side chains were visible, including most aromatic residues and arginines). During refinement, torsion angle, planar peptide, and Ramachandran restraints were used in order not to create a well-fitting but unrealistic model. The final model contains 878 residues out of a possible 903, a [NiFe] cluster, 4 [4Fe4S] clusters, an FAD and part of an F 420 molecule; 88.1% of residues lie in the most favored regions of a Ramachandran plot, 8.0% in generously allowed regions, and 3.8% are outliers. A full dodecamer model was generated in Chimera ( Pettersen et al., 2004 ) and a map was calculated from this model. An FSC curve between this map and the experimental map indicated an overall agreement of 5.8 Å at 0.5 FSC ( Figure 10C ). Figures were made using Chimera ( Pettersen et al., 2004 ) and the PyMOL Molecular Graphics System ( DeLano, 2002 ).

Additional files Major datasets

The following datasets were generated: Mills DJ , Vitt S , Strauss M , Shima S , Vonck J , 2013 , F420-reducing [NiFe] hydrogenase Frh , EMD-2096 ; http://www.ebi.ac.uk/pdbe-srv/emsearch/atlas/2096_summary.html , Publicly available at the Electron Microscopy Data Bank ( http://www.ebi.ac.uk/pdbe/emdb/ ). Mills DJ , Vitt S , Strauss M , Shima S , Vonck J , 2013 , F420-reducing [NiFe] hydrogenase Frh with bound substrate , EMD-2097 ; http://www.ebi.ac.uk/pdbe-srv/emsearch/atlas/2097_summary.html , Publicly available at the Electron Microscopy Data Bank ( http://www.ebi.ac.uk/pdbe/emdb/ ). Mills DJ , Vitt S , Strauss M , Shima S , Vonck J , 2013 , Cryo-EM structure of the F420-reducing NiFe-hydrogenase from a methanogenic archaeon with bound substrate , 3zfs ; http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3zfs , Publicly available at the RCSB Protein Data Bank ( http://www.rcsb.org/pdb/ ). The following previously published datasets were used: Liesegang H , Kaster A-K , Wiezer A , Goenrich M , Wollherr A , Seedorf H , Gottschalk G , Thauer RK , 2010 , Complete Genome Sequence of Methanothermobacter marburgensis, a Methanoarchaeon Model Organism , CP001710 ; http://www.ncbi.nlm.nih.gov/nuccore/CP001710 , Publicly available at GenBank. Adman ET , Sieker LC , 2000 , The 2[4Fe-4S] Ferredoxins , 1dur ; http://www.rcsb.org/pdb/explore/explore.do?structureId=1dur , Publicly available at the RCSB Protein Data Bank ( http://www.rcsb.org/pdb/ ). Marques MC , Coelho R , De Lacey AL , Pereira IAC , Matias PM , 2010 , The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, “as-isolated” state. , 2wpn ; http://www.rcsb.org/pdb/explore/explore.do?structureId=2wpn , Publicly available at the RCSB Protein Data Bank ( http://www.rcsb.org/pdb/ ).