Semi‐automated protein crystal mounting device for the sulfur single‐wavelength anomalous diffraction method

Use of longer-wavelength X-rays has advantages for the detection of small anomalous signals from light atoms, such as sulfur, in protein molecules. However, the accuracy of the measured diffraction data decreases at longer wavelengths because of the greater X-ray absorption. The capillary-top mounting method (formerly the loopless mounting method) makes it possible to eliminate frozen solution around the protein crystal and reduces systematic errors in the evaluation of small anomalous differences. However, use of this method requires custom-made tools and a large amount of skill. Here, the development of a device that can freeze the protein crystal semi-automatically using the capillary-top mounting method is described. This device can pick up the protein crystal from the crystallization drop using a micro-manipulator, and further procedures, such as withdrawal of the solution around the crystal by suction and subsequent flash freezing of the protein crystal, are carried out automatically. This device makes it easy for structural biologists to use the capillary-top mounting method for sulfur single-wavelength anomalous diffraction phasing using longer-wavelength X-rays.

[1]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[2]  A. Wlodawer,et al.  Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed* , 2009, The Journal of Biological Chemistry.

[3]  A. McPherson,et al.  Structures of three crystal forms of the sweet protein thaumatin. , 1994, Acta crystallographica. Section D, Biological crystallography.

[4]  U. Mueller,et al.  De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse. , 2009, Acta crystallographica. Section D, Biological crystallography.

[5]  N. Chayen,et al.  Structure of lobster apocrustacyanin A1 using softer X-rays. , 2001, Acta crystallographica. Section D, Biological crystallography.

[6]  B. C. Wang.  Resolution of phase ambiguity in macromolecular crystallography. , 1985, Methods in enzymology.

[7]  Florent Cipriani,et al.  C3D: a program for the automated centring of cryocooled crystals. , 2006, Acta crystallographica. Section D, Biological crystallography.

[8]  G Bricogne,et al.  Can anomalous signal of sulfur become a tool for solving protein crystal structures? , 1999, Journal of molecular biology.

[9]  G. Sheldrick,et al.  In-house phase determination of the lima bean trypsin inhibitor: a low-resolution sulfur-SAD case. , 2003, Acta crystallographica. Section D, Biological crystallography.

[10]  J. Helliwell,et al.  The interdependence of wavelength, redundancy and dose in sulfur SAD experiments. , 2008, Acta crystallographica. Section D, Biological crystallography.

[11]  Alexander McPherson,et al.  Operator-assisted harvesting of protein crystals using a universal micromanipulation robot , 2007, Journal of applied crystallography.

[13]  W. Hunter,et al.  De novo phasing of two crystal forms of tryparedoxin II using the anomalous scattering from S atoms: a combination of small signal and medium resolution reveals this to be a general tool for solving protein crystal structures. , 2002, Acta crystallographica. Section D, Biological crystallography.

[14]  E J Gordon,et al.  The C1 subunit of alpha-crustacyanin: the de novo phasing of the crystal structure of a 40 kDa homodimeric protein using the anomalous scattering from S atoms combined with direct methods. , 2001, Acta crystallographica. Section D, Biological crystallography.

[15]  C. Cambillau,et al.  Sulfur Single-wavelength Anomalous Diffraction Crystal Structure of a Pheromone-Binding Protein from the Honeybee Apis mellifera L , 2003 .

[16]  齐建勋,et al.  An improved loopless mounting method for cryocrystallography , 2010 .

[17]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[18]  D. Velmurugan,et al.  A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A2 at 1.6 A resolution using sulfur-SAS at 1.54 A wavelength. , 2004, Acta crystallographica. Section D, Biological crystallography.

[19]  N. Watanabe.  From phasing to structure refinement in-house: Cr/Cu dual-wavelength system and a loopless free crystal-mounting method. , 2006, Acta crystallographica. Section D, Biological crystallography.

[20]  M. Weiss,et al.  Soft X-rays, high redundancy, and proper scaling: a new procedure for automated protein structure determination via SAS. , 2001, Structure.

[21]  Didier Nurizzo,et al.  An inexpensive automatically operated device for the flash annealing of crystals of macromolecules , 2009 .

[22]  J. Rose,et al.  Structure of the Ca2+‐regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur substructure , 2000, Protein science : a publication of the Protein Society.

[23]  Robert E. Thorne,et al.  Microfabricated mounts for high-throughput macromolecular cryocrystallography , 2003 .

[24]  M. Weiss,et al.  On the routine use of soft X-rays in macromolecular crystallography. , 2001, Acta crystallographica. Section D, Biological crystallography.

[25]  D. Bourgeois,et al.  Structural basis for the phototoxicity of the fluorescent protein KillerRed , 2009, FEBS letters.

[26]  C. Wilmot,et al.  Synergy within structural biology of single crystal optical spectroscopy and X-ray crystallography. , 2007, Current opinion in structural biology.

[27]  J. Pflugrath,et al.  Applications of anomalous scattering from S atoms for improved phasing of protein diffraction data collected at Cu Kalpha wavelength. , 2001, Acta crystallographica. Section D, Biological crystallography.

[28]  I. Tanaka,et al.  Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron* , 2008, Journal of Biological Chemistry.

[29]  G. Sheldrick,et al.  In-house measurement of the sulfur anomalous signal and its use for phasing. , 2003, Acta crystallographica. Section D, Biological crystallography.

[30]  Anastassis Perrakis,et al.  Automated protein model building combined with iterative structure refinement , 1999, Nature Structural Biology.

[31]  Z. Dauter,et al.  Phasing on anomalous signal of sulfurs: what is the limit? , 2003, Acta crystallographica. Section D, Biological crystallography.

[32]  R. Thorne,et al.  Slow cooling of protein crystals. , 2009, Journal of applied crystallography.

[33]  I. Tanaka,et al.  Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method. , 2005, Acta crystallographica. Section D, Biological crystallography.

[34]  Wayne A. Hendrickson,et al.  Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur , 1981, Nature.

[35]  W. Hunter,et al.  Structure of the macrocycle thiostrepton solved using the anomalous dispersion contribution of sulfur. , 2001, Acta crystallographica. Section D, Biological crystallography.

[36]  R. Esnouf,et al.  Structure of a functional IGF2R fragment determined from the anomalous scattering of sulfur , 2002, The EMBO journal.

[37]  I. Tanaka,et al.  Structural basis of CoA recognition by the Pyrococcus single-domain CoA-binding proteins , 2007, Journal of Structural and Functional Genomics.

[38]  G. Sheldrick.  A short history of SHELX. , 2008, Acta crystallographica. Section A, Foundations of crystallography.

[39]  J P Abrahams,et al.  Matrix methods for solving protein substructures of chlorine and sulfur from anomalous data. , 2001, Acta crystallographica. Section D, Biological crystallography.

[40]  Ming Luo,et al.  Crystal Structure of the Cytoskeleton-associated Protein Glycine-rich (CAP-Gly) Domain* , 2002, The Journal of Biological Chemistry.

[41]  I. Tanaka,et al.  Comparison of phasing methods for sulfur-SAD using in-house chromium radiation: case studies for standard proteins and a 69 kDa protein. , 2005, Acta crystallographica. Section D, Biological crystallography.

[42]  T. Teng,et al.  Mounting of crystals for macromolecular crystallography in a free-standing thin film , 1990 .