Volume 25, Issue 9, Pages 1461-1468.e2 (September 2017) From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography  Robert Dods, Petra Båth, David Arnlund, Kenneth R. Beyerlein, Garrett Nelson, Mengling Liang, Rajiv Harimoorthy, Peter Berntsen, Erik Malmerberg, Linda Johansson, Rebecka Andersson, Robert Bosman, Sergio Carbajo, Elin Claesson, Chelsie E. Conrad, Peter Dahl, Greger Hammarin, Mark S. Hunter, Chufeng Li, Stella Lisova, Despina Milathianaki, Joseph Robinson, Cecilia Safari, Amit Sharma, Garth Williams, Cecilia Wickstrand, Oleksandr Yefanov, Jan Davidsson, Daniel P. DePonte, Anton Barty, Gisela Brändén, Richard Neutze  Structure  Volume 25, Issue 9, Pages 1461-1468.e2 (September 2017) DOI: 10.1016/j.str.2017.07.002 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1461-1468.e2DOI: (10.1016/j.str.2017.07.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Crystals of the Blastochloris viridis Photosynthetic Reaction Center (A) Macrocrystals. (B) Crushed macrocrystals used to collect the 2014 dataset. (C) Microcrystals recovered after one round of seeding used to collect the 2015 dataset. (D) Microcrystals recovered after two rounds of seeding used to collect the 2016 dataset. Structure 2017 25, 1461-1468.e2DOI: (10.1016/j.str.2017.07.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Improvements in Electron Density Deriving from SFX Data Collected at Room Temperature Electron density is shown for the flexible Glu45-Pro54 region of the H subunit. 2Fo-Fc (gray) and Fo-Fc (green) electron density maps are contoured at 1σ and 3σ, respectively. (A) Electron density calculated to 1.9-Å resolution from diffraction data collected using conventional cryo-crystallography. This structural model (PDB: 2I5N) was used for molecular replacement in this work and the electron density maps were calculated from structure factors deposited in the PDB. (B) 2Fo-Fc and Fo-Fc electron density map calculated from XFEL data before the loop is modeled such that no phase information is used for this region (an omit map). (C) 2Fo-Fc electron density map showing continuous electron density calculated after modeling the main chain of this loop. Structure 2017 25, 1461-1468.e2DOI: (10.1016/j.str.2017.07.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Lipid Molecules Visible in the SFX Structure RCvir Electron density (gray) and modeled lipids (black) are superimposed upon lipid molecules found in structures of RCvir determined using synchrotron data (colored: PDB: 1DXR, blue; 6PRC, pink; 1R2C, cyan; 2I5N, red). SFX electron density maps (gray) are contoured at 1σ. Structure 2017 25, 1461-1468.e2DOI: (10.1016/j.str.2017.07.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Tree Diagram Representing Structural Similarities across All Deposited RCvir Structures Clustering was performed by comparing the average of the internal difference matrix calculated on Cα atoms (Wickstrand et al., 2015). The SFX structures (PDB: 5O64, 5O4C, and 5NJ4) cluster with synchrotron structures of the same space group. 5O64 refers to the structure obtained from crushed macrocrystals, 5O4C refers to the structure obtained from crystals after one round of seeding, and 5NJ4 refers to the structure from crystals obtained after two rounds of seeding. Structure 2017 25, 1461-1468.e2DOI: (10.1016/j.str.2017.07.002) Copyright © 2017 Elsevier Ltd Terms and Conditions