Protein Crystallography

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His6or a dual His6-MBP tagged fusion protein by Gateway® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His6 tag or a His6-MBP tag can be made on the basis of this solubility test.

[1]  C. Darwin.  XXXIV. The theory of X-ray reflexion , 1914 .

[2]  J. Kendrew,et al.  A Three-Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis , 1958, Nature.

[3]  M. Perutz,et al.  Structure of Hæmoglobin: A Three-Dimensional Fourier Synthesis at 5.5-Å. Resolution, Obtained by X-Ray Analysis , 1960, Nature.

[4]  R. Crowther,et al.  A computer-linked cathode-ray tube microdensitometer for x-ray crystallography. , 1968, Journal of scientific instruments.

[5]  K. Lonsdale.  X-Ray Diffraction , 1971, Nature.

[6]  The use of rotation and translation functions in the interpretation of low resolution electron density maps. , 1976, Journal of molecular biology.

[7]  U. W. Arndt,et al.  The Rotation method in crystallography : data collection from macromolecular crystals , 1977 .

[8]  S. Harrison,et al.  Tomato bushy stunt virus at 2.9 Å resolution , 1978, Nature.

[9]  H. Hope.  Cryocrystallography of biological macromolecules: a generally applicable method. , 1988, Acta crystallographica. Section B, Structural science.

[10]  Richard Henderson,et al.  Cryo-protection of protein crystals against radiation damage in electron and X-ray diffraction , 1990, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[11]  A. Brünger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures , 1992, Nature.

[12]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[13]  Manfred Burghammer,et al.  Small is beautiful: protein micro-crystallography , 1998, Nature Structural Biology.

[14]  D. Cruickshank,et al.  Remarks about protein structure precision. , 1999, Acta crystallographica. Section D, Biological crystallography.

[15]  Z Dauter,et al.  Data-collection strategies. , 1999, Acta crystallographica. Section D, Biological crystallography.

[16]  W A Hendrickson,et al.  Synchrotron crystallography. , 2000, Trends in biochemical sciences.

[17]  M G Rossmann.  Molecular replacement--historical background. , 2001, Acta crystallographica. Section D, Biological crystallography.

[18]  A. Vagin,et al.  Spherically averaged phased translation function and its application to the search for molecules and fragments in electron-density maps. , 2001, Acta crystallographica. Section D, Biological crystallography.

[19]  D. Blow,et al.  Rearrangement of Cruickshank's formulae for the diffraction-component precision index. , 2002, Acta crystallographica. Section D, Biological crystallography.

[20]  J. Tanner,et al.  MRSAD: using anomalous dispersion from S atoms collected at Cu Kalpha wavelength in molecular-replacement structure determination. , 2003, Acta crystallographica. Section D, Biological crystallography.

[21]  S. Harrison,et al.  How does radiation damage in protein crystals depend on X-ray dose? , 2003, Structure.

[22]  Wladek Minor,et al.  Measurement errors and their consequences in protein crystallography. , 2003, Acta crystallographica. Section D, Biological crystallography.

[23]  John Mongan,et al.  Interactive essential dynamics , 2004, J. Comput. Aided Mol. Des..

[24]  Philippe Carpentier,et al.  Automated analysis of vapor diffusion crystallization drops with an X-ray beam. , 2004, Structure.

[25]  Elspeth F Garman,et al.  Experimental determination of the radiation dose limit for cryocooled protein crystals. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Keiko Ikeda,et al.  The molecular organization of cypovirus polyhedra , 2007, Nature.

[27]  K. Schmidt,et al.  Gas dynamic virtual nozzle for generation of microscopic droplet streams , 2008, 0803.4181.

[28]  Wilhelm Pfleging,et al.  Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis. , 2009, Lab on a chip.

[29]  James M. Holton,et al.  A beginner’s guide to radiation damage , 2009, Journal of synchrotron radiation.

[30]  R. Stevens,et al.  Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam , 2009, Journal of The Royal Society Interface.

[31]  D. Stuart,et al.  How baculovirus polyhedra fit square pegs into round holes to robustly package viruses , 2010, The EMBO journal.

[32]  Henry N. Chapman,et al.  Femtosecond X-ray protein nanocrystallography , 2010 .

[33]  Wolfgang Kabsch,et al.  Integration, scaling, space-group assignment and post-refinement , 2010, Acta crystallographica. Section D, Biological crystallography.

[34]  George M. Sheldrick,et al.  Experimental phasing with SHELXC/D/E: combining chain tracing with density modification , 2010, Acta crystallographica. Section D, Biological crystallography.

[35]  R. Ismagilov,et al.  Protein crystallization using microfluidic technologies based on valves, droplets, and SlipChip. , 2010, Annual review of biophysics.

[36]  Kenneth A. Frankel,et al.  The minimum crystal size needed for a complete diffraction data set , 2010, Acta crystallographica. Section D, Biological crystallography.

[37]  Nathaniel Echols,et al.  Accessing protein conformational ensembles using room-temperature X-ray crystallography , 2011, Proceedings of the National Academy of Sciences.

[38]  T. Tomizaki,et al.  SLS Crystallization Platform at Beamline X06DA—A Fully Automated Pipeline Enabling in Situ X-ray Diffraction Screening , 2011 .

[39]  Lirong Chen,et al.  A multi-dataset data-collection strategy produces better diffraction data , 2011, Acta crystallographica. Section A, Foundations of crystallography.

[40]  Marcus Mueller,et al.  Optimal fine ϕ slicing for single photon counting pixel detectors , 2011 .

[41]  G. Evans,et al.  The design of macromolecular crystallography diffraction experiments , 2011, Acta crystallographica. Section D, Biological crystallography.

[42]  E. Pai,et al.  X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection , 2011, Acta crystallographica. Section D, Biological crystallography.

[43]  Sandor Brockhauser,et al.  Translation calibration of inverse-kappa goniometers in macromolecular crystallography , 2011, Acta crystallographica. Section A, Foundations of crystallography.

[44]  Frank von Delft,et al.  Assessment of radiation damage behaviour in a large collection of empirically optimized datasets highlights the importance of unmeasured complicating effects , 2011, Journal of synchrotron radiation.

[45]  Gwyndaf Evans,et al.  In situ macromolecular crystallography using microbeams , 2012, Acta crystallographica. Section D, Biological crystallography.

[46]  Masaki Yamamoto,et al.  Micro-crystallography comes of age. , 2012, Current opinion in structural biology.

[47]  Sébastien Boutet,et al.  Nanoflow electrospinning serial femtosecond crystallography. , 2012, Acta crystallographica. Section D, Biological crystallography.

[48]  Julia Brasch,et al.  Structures from Anomalous Diffraction of Native Biological Macromolecules , 2012, Science.

[49]  P. Andrew Karplus,et al.  Linking Crystallographic Model and Data Quality , 2012, Science.

[50]  Garth J. Williams,et al.  High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography , 2012, Science.

[51]  Georg Weidenspointner,et al.  Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements , 2011, Nature Photonics.

[52]  Florent Cipriani,et al.  CrystalDirect: a new method for automated crystal harvesting based on laser-induced photoablation of thin films. , 2012, Acta crystallographica. Section D, Biological crystallography.

[53]  K. Diederichs,et al.  Better models by discarding data? , 2013, Acta crystallographica. Section D, Biological crystallography.

[54]  Chipx: A Novel Microfluidic Chip for Counter- Diffusion Crystallization of Biomolecules and in Situ Crystal Analysis at Room Temperature , 2013 .

[55]  Jennifer L Wierman,et al.  Graphene as a protein crystal mounting material to reduce background scatter. , 2013, Journal of applied crystallography.

[56]  Alexei S Soares,et al.  Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines. , 2013, Journal of synchrotron radiation.

[57]  R. Sweet,et al.  Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt , 2014, Journal of synchrotron radiation.

[58]  Anton Barty,et al.  Fixed-target protein serial microcrystallography with an x-ray free electron laser , 2014, Scientific Reports.

[59]  Yiping Feng,et al.  Goniometer-based femtosecond crystallography with X-ray free electron lasers , 2014, Proceedings of the National Academy of Sciences.

[60]  Fei Long,et al.  The PDB_REDO server for macromolecular structure model optimization , 2014, IUCrJ.

[61]  Kunio Hirata,et al.  Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL , 2014, Nature Methods.

[62]  Michael Heymann,et al.  Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction , 2014, IUCrJ.

[63]  Anton Barty,et al.  Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser , 2014, Nature.

[64]  Anton Barty,et al.  Room-temperature macromolecular serial crystallography using synchrotron radiation , 2014, IUCrJ.

[65]  Wei Liu,et al.  Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography , 2014, Nature Protocols.

[66]  Anton Barty,et al.  Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography , 2014, Nature Communications.

[67]  D. Stuart,et al.  Exploiting fast detectors to enter a new dimension in room-temperature crystallography , 2014, Acta crystallographica. Section D, Biological crystallography.

[68]  Kay Diederichs,et al.  Breaking the indexing ambiguity in serial crystallography. , 2014, Acta crystallographica. Section D, Biological crystallography.

[69]  Florence Tama,et al.  Hybrid Electron Microscopy Normal Mode Analysis graphical interface and protocol. , 2014, Journal of structural biology.

[70]  D. Stuart,et al.  In cellulo structure determination of a novel cypovirus polyhedrin , 2014, Acta crystallographica. Section D, Biological crystallography.

[71]  Henry N. Chapman,et al.  Serial crystallography on in vivo grown microcrystals using synchrotron radiation , 2014, IUCrJ.

[72]  Garth J. Williams,et al.  Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser , 2014, Nature.

[73]  A. N. Popov,et al.  MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines , 2015, Acta crystallographica. Section D, Biological crystallography.

[74]  J. Berger,et al.  A high-transparency, micro-patternable chip for X-ray diffraction analysis of microcrystals under native growth conditions , 2015, Acta crystallographica. Section D, Biological crystallography.

[75]  Yoshiki Tanaka,et al.  Grease matrix as a versatile carrier of proteins for serial crystallography , 2014, Nature Methods.

[76]  Aaron S. Brewster,et al.  Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams , 2015, Acta crystallographica. Section D, Biological crystallography.

[77]  Identification of rogue datasets in serial crystallography , 2015 .

[78]  Jesse B. Hopkins,et al.  Figures and figure supplements Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography , 2016 .

[79]  C. Schulze-Briese,et al.  PRIGo: a new multi-axis goniometer for macromolecular crystallography , 2015, Journal of synchrotron radiation.

[80]  W. Hendrickson,et al.  Crystallographic phasing from weak anomalous signals. , 2015, Current opinion in structural biology.

[81]  C. David,et al.  A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering , 2015, Scientific Reports.

[82]  Sébastien Boutet,et al.  A novel inert crystal delivery medium for serial femtosecond crystallography , 2015, IUCrJ.

[83]  Ilme Schlichting,et al.  Serial femtosecond crystallography: the first five years , 2015, IUCrJ.

[84]  Ezequiel Panepucci,et al.  In meso in situ serial X-ray crystallography of soluble and membrane proteins , 2015, Acta crystallographica. Section D, Biological crystallography.

[85]  Sébastien Boutet,et al.  Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography , 2015, Cell.

[86]  S. Iwata,et al.  Structure determination of an integral membrane protein at room temperature from crystals in situ , 2015, Acta crystallographica. Section D, Biological crystallography.

[87]  Anton Barty,et al.  Structural basis for bifunctional peptide recognition at human δ-Opioid receptor , 2015, Nature Structural &Molecular Biology.

[88]  Kay Diederichs,et al.  Assessing and maximizing data quality in macromolecular crystallography. , 2015, Current opinion in structural biology.

[89]  Martin Caffrey,et al.  A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes , 2015, Acta crystallographica. Section F, Structural biology communications.

[90]  Nicholas K. Sauter,et al.  Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array , 2015, Acta crystallographica. Section D, Biological crystallography.

[91]  Anton Barty,et al.  Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams. , 2014, Journal of synchrotron radiation.

[92]  Kanagaraj Sekar,et al.  Online_DPI: a web server to calculate the diffraction precision index for a protein structure , 2015 .

[93]  G. Evans,et al.  In vacuo X-ray data collection from graphene-wrapped protein crystals , 2015, Acta crystallographica. Section D, Biological crystallography.

[94]  Ezequiel Panepucci,et al.  Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams. , 2015, Acta crystallographica. Section D, Biological crystallography.

[95]  Manfred Burghammer,et al.  Lipidic cubic phase serial millisecond crystallography using synchrotron radiation , 2015, IUCrJ.

[96]  Ezequiel Panepucci,et al.  Fast native-SAD phasing for routine macromolecular structure determination , 2014, Nature Methods.

[97]  G. Labesse,et al.  Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography. , 2015, Acta crystallographica. Section D, Biological crystallography.

[98]  Veit Elser,et al.  Determination of crystallographic intensities from sparse data , 2015, IUCrJ.

[99]  M. Steinmetz,et al.  Data-collection strategy for challenging native SAD phasing , 2016, Acta crystallographica. Section D, Structural biology.

[100]  J. A. Gavira,et al.  Current trends in protein crystallization. , 2016, Archives of biochemistry and biophysics.

[101]  Jessica D. Schiffman,et al.  Graphene-based microfluidics for serial crystallography. , 2016, Lab on a chip.

[102]  Takashi Tomizaki,et al.  Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature , 2016, Scientific Reports.

[103]  Arnaud Hungler,et al.  ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants , 2016, Journal of applied crystallography.

[104]  Przemyslaw J Porebski,et al.  Protein purification and crystallization artifacts: The tale usually not told , 2016, Protein science : a publication of the Protein Society.

[105]  Peter Murphy,et al.  Automated harvesting and processing of protein crystals through laser photoablation , 2016, Acta crystallographica. Section D, Structural biology.

[106]  Applications of thin-film sandwich crystallization platforms , 2016, Acta crystallographica. Section F, Structural biology communications.

[107]  W. DeGrado,et al.  High-density grids for efficient data collection from multiple crystals , 2016, Acta crystallographica. Section D, Structural biology.

[108]  Marion Boudes,et al.  A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction , 2016, Acta crystallographica. Section D, Structural biology.

[109]  K. Diederichs,et al.  In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures , 2016, Acta crystallographica. Section D, Structural biology.

[110]  A. Kuczewski,et al.  Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography. , 2016, Structure.

[111]  Vadim Cherezov,et al.  Serial Femtosecond Crystallography of G Protein-Coupled Receptors. , 2018, Annual review of biophysics.