Crystallography

All RC maquette crystals were grown in hanging drops at 4 °C. Protein stock solutions contained 20–40 mM NaCl and 10–20 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer at pH 6.5 and were stored at 4 °C. Small amounts of porphyrin cofactors were added at room temperature from dimethyl sulfoxide stock solutions until a 1.0:1.0 equimolar ratio of porphyrin to protein was reached. Several minutes of equilibration time were given between additions of porphyrin in order to prevent aggregation and precipitation of cofactor. (Heme B was added prior to ZnP for crystal structure in PDB ID: 5VJS). Sample buffers were exchanged to the low salt PIPES buffer using 5 kDa MWCO Vivaspin Turbo 15 concentrators (Sartorius AG). Supplementary Table ⁴ gives for each crystal the RC maquette stock concentration used, cofactors included in the stock solution, well solution composition, drop volume, and cryoprotectant for each crystal structure. Crystal structure 5VJU used streak seeding with a cat whisker.

All X-ray crystallographic data sets were collected from single crystals at 100 K. X-ray diffraction data for crystal structures with PDB IDs 5VJS and 5VJT were collected at the National Synchrotron Light Source, beamline X6A using an ADSC Q270 CCD x-ray area detector. X-ray diffraction data for the crystal structure of L71H mutant (PDB ID: 5VJU) was collected using a Rigaku Micromax-007 HF rotating copper anode X-ray generator and VariMax HF optics with a Rigaku Saturn 944 HG CCD detector. Multiwavelength anomalous dispersion (MAD) data was collected to solve crystal structure 5VJS. Data were integrated with XDS⁵⁶ and initial phases were calculated using SOLVE⁵⁷. All other crystals used the 5VJS crystal structure as a model for molecular replacement using Phaser⁵⁸. Software packages CCP4i⁵⁹ and PHENIX^60,61 were used throughout the structure solution and refinement process. Intensities were scaled using SCALA⁶². Refinement was done using REFMAC5⁶³, phenix.refine⁶⁴, and PDB_REDO⁶⁵. Real space refinement was done by manually fitting models into electron density maps in Coot⁶⁶. Density modification was done using RESOLVE⁶⁷. Composite omit maps were created with simulated annealing in PHENIX to reduce model bias^64,68,69. Omit maps of cofactor binding sites are presented in Supplementary Fig. ². Polder omit maps in Supplementary Fig. ¹³ were also created in PHENIX⁷⁰. Statistics for the three data sets collected for MAD solution of crystal structure 5VJS are given in Supplementary Table ⁵. Data collection and refinement statistics for all three crystal structures, 5VJS, 5VJT, and 5VJU, are given in Supplementary Table ⁶. Protein visualization and preparation of X-ray crystal structure images for figures was done in The PyMOL Molecular Graphics System, Version 2.5.0a0, Schrödinger, LLC.