References Rpd
1. Allen, F. H. 2002 The Cambridge structural database a quarter of a million crystal structures and rising. Acta Cryst. B58, 380-388. 2. K nig, V., Vertesy, L., and Schneider, T. R. 2003 Structure of the a-amylase inhibitor tendamistat at 0.93 . Acta Cryst. D59,1737-1743. 3. Diamond, R. 1971 A real-space refinement procedure for proteins. Acta Cryst. A27, 436-452. 4. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., et al. 1977 The Protein Data Bank a computer-based archival file for...
Notes Nsd
1. At room temperature few protein crystals will survive longer than a minute of exposure to synchrotron radiation, and although radiation from a home source is much less intense, the increased duration of the experiment is also likely to result in severe damage to an unprotected crystal see Chapter 1 on cryocooling . 2. The majority of the data reduction programs calculate the mean amplitude, lt Fh gt 1 n I Fi , and the standard deviation of the mean, o Fh 1 n n - 1 I Fi - Fh 2 1 2. As n...
Phasing
In general, the structure solution of diffraction data from halide-soaked crystals may proceed along well-established protocols for utilization of the anomalous signal in MAD, single-wavelength anomalous diffraction SAD , or MIRAS techniques, and any of the suitable programs can be used. However, in contrast to the popular MAD approach based on selenomethionines, the number of identifiable anomalous scatterer sites cannot be predicted. The halide-soaked crystals always have a large number of...
SnB
A complete description of the SnB algorithm as implemented in the direct methods program, SnB, has been published 18,27 see also the website, http www.hwi.buffalo.edu SnB and will not be described in any detail here. The first thing that is required when running SnB is to create a configuration file, which is accomplished by invoking the General Information menu. Information such as unit cell constants, unit cell contents see Note 7 , and other information are entered here. Once the general...
The Beam Position Relative to the Rotation AxisAxes
The goniometer rotation axis axes must intersect with the beam path and with the line of sight of the crystal viewing alignment camera. The monitor attached to the camera will show a set of cross wires, and it can be dangerous to assume that if the crystal is placed there, the beam will hit it. In-house you can check the alignment by mounting a metal pin on the goniometer head, removing the backstop only do this test under the supervision of the person responsible for the X-ray equipment and...
U
P u is the Patterson function at point u. Pr ur is the rotated version. U is the volume of the sphere of the Patterson contained by the radius of integration. Rotation angles that bring the Patterson maps into coincidence high value of integration correspond to peaks in the rotation function map. This map is conveniently plotted in sections of k. For example, peaks on the k 180 section indicate the presence of all the twofold rotation and screw axes, peaks on the k 120 section indicate the...
Rt3
We have designed a helium cryostream that consists of a liquid helium stream for cooling the crystal and an exterior, concentric flow of warm, room-temperature gas see Note 1 . The gas flow rate of the inner stream is controlled by the needle valve of a liquid helium transfer line Janis see Note 2 . The regulator from a cylinder of helium gas controls the gas flow rate of the outer stream see Note 3 . Recently, cryostreams from commercial suppliers have become available. These ultra-low...
Elspeth Garman and Robert M Sweet
Instruments, methods, and software for modern macromolecular crystallography is becoming so effective that molecular biologists often can solve structures from their crystals by working without a license. In this chapter, the authors attempt to demystify some of the apparatus and techniques by providing a roadmap. Current methods for collecting X-ray diffraction data from macromolecular crystals are described. The principles of operation of the required X-ray sources, optics, goniometers, and...
Conditions for Merohedral Twinning
Not all space groups are prone to twinning, and so not all crystals require the application of twinning tests. The possibility of twinning requires certain relationships among cell parameters. In general, merohedral twinning can occur whenever the rotational symmetry of the lattice exceeds that of the underlying crystal point group 20 . Take for example a P4 crystal the tetragonal lattice contains, in addition to the crystallographic fourfold, a perpendicular twofold symmetry that exceeds the...
The DetectortoCrystal Distance Resolution
The closer the detector is to the crystal, the higher the resolution of the data that can be collected. The relationship is illustrated in Fig. 14 and is calculated using basic trigonometry and Bragg's law see Fig. 14 legend . Modern data collection software usually tells you the resolution at a specific detector distance, but it is often useful to be able to calculate it yourself. The resolution you will need for your experiment depends critically on what is the problem being addressed for...
B Aaw
Bond lengths, 232-234, 237-238, 249, 256, 266 Bragg's law, 46 Bravais lattices, 46-47 Bromides, 149 Carbon monoxide, 22 CCD-based detector, 73 CCP4 suite of programs , 128, 257 Cell parameters, 44 X2 chi-square , 90 Chloride, 149 CHOOCH program , 227 CNS program , 114, 134, 208, 251 Cold stream, 32 Collimation, 84 Collimator, 64 Combinatorial libraries, 174 COMO program , 131 Competition assay, 160 Completeness, 80 Computer programs also see individual computer names , 273-278 Conjugate...
Stephen J Everse and Sylvie Doublie
Virtually all software is constantly changing and evolving and crystallographic software is no exception , which makes it nearly impossible to write a chapter that will remain current. In this chapter, we introduce CRYSTAL, a website http crystal.uvm.edu where a comprehensive list of available crystallographic packages for each step of macromolecular structure determination will be maintained. Additionally, we provide links to books, journals, and a number of educational sites on...
Substructure Refinement and Completion
All programs currently in use for the determination of substructures will produce a model for the substructure that has the best agreement with a given set of substructure structure factors. However, for the determination of the best phases on which the initial electron density map for the complete structure will be based, it may be advantageous to use a substructure model that is consistent with all experimental data. For instance, in MAD phasing, the substructure should not only be in...
Torsion Angle Parameterization
The substitution of atomic coordinates with torsion angles dramatically reduces the total number of parameters see Fig. 1 . This decrease is advantageous when the resolution of the diffraction data is quite low lower than 3 A . At these resolutions there are many fewer reflections to define the values of parameters in the traditional model. Even with the addition of bond length and angle information as restraints, these models tend to get stuck in local minima or overfit the data. Increasing...
Mounting Macromolecular Crystals for XRay Diffraction
There are two methods to mount crystals for X-ray analysis, either in thin-walled capillaries glass or quartz or in fiber loops. Capillary-mounted crystals are currently only used for preliminary analyses or for recording intensity data from those samples not amenable to shock cooling. Measurements of X-ray diffraction at cryogenic temperatures are routinely performed using shock-cooled crystals that have been mounted in thin films supported by fiber loops. Both types of mounts will be...
Estimation of Substructure Structure Factors
Because the number of atoms in a native macromolecular structure is usually much larger than the number of additional heavy atoms in a derivative, it is a valid approximation to assume FH lt lt FPH, where FH are the structure factor amplitudes corresponding to the substructure only, and FPH the structure factor amplitudes of the derivative. This approximation leads to 2 , Subheading 6.2. therein Fh lt lt Fph Fph - Fp Fh cosiypH - Wh 1 The cosine term takes on values between -1 and 1. Therefore...
Estimating the Number of Molecules in the Asymmetric Unit With VM
The number of molecules in the asymmetric unit n can be readily estimated using a method developed by Matthews 2 . His survey of 116 protein crystals revealed that the v m ratio of the asymmetric unit VM typically ranges between 2.0 to 3.0 A3 Da corresponding to a solvent content of 40-60 by volume . He hypothesized that this range of VM is characteristic of the majority of protein crystals and, thus, could be used as a guide to estimate n for any protein crystal given its unit cell volume and...
HeavyAtom Derivatization for Phasing
Strategies abound for choosing among the traditional heavy-atom derivatiz-ing agents 1,2 . Conditions that have yielded successful derivatization have also been compiled 3-5 . Although in the past it had been difficult and expensive to amass a collection of derivatizing agents, kits are now commercially available that contain small quantities of a broad sampling of historically useful compounds. Rather than recapitulating the strategies for ranking these compounds for particular cases, the...
References Gzz
1. Green, D. W., Ingram, V. M., and Perutz, M. F. 1954 The Structure of hemoglobin. IV Sign determination by the isomorphous replacement method. Proc. R. Soc. London Ser. A 225, 287-307. 2. Hendrickson, W. A. and Teeter, M. M. 1981 Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur. Nature London 290, 107-113. 3. Hendrickson, W. A., Horton, J. R. and LeMaster, D. M. 1990 Selenomethionyl proteins produced for analysis of multiwavelength...
Info Qkc
the uncombined one, but it is more seamless. COMO does away with the idea that correct orientations will have a high rotation function score and instead subscribes to the notion that the best assessment of an orientation is by its behavior in the translation function. Therefore, multiple rotation angles number specified by the user are automatically checked in the translation function calculation until the correct solution is hopefully found. The rotation function used is the Crowther fast...
Maximum Likelihood Molecular Replacement in Phaser
The most likely model is one that maximizes the probability that the experimental observations were made. This simple statement is the overriding principle behind maximum likelihood. Over the past 10 yr, the statistical method of maximum likelihood has come to dominate most aspects of struc ture determination, most prominently experimental phasing and structure refinement. As the success of these efforts became apparent, the idea of using maximum likelihood to improve the sensitivity of...
Improving Data Quality
One of the most important issues in producing good data is the quality of the beam-collimation system. The importance of this is reflected in the fact that it is Fig. 15. A typical collimation system. The size of the beam is determined by the limiting aperture. The guard aperture catches scatter from the limiting aperture. The beamstop should capture all of this scatter as well as the main beam. Fig. 15. A typical collimation system. The size of the beam is determined by the limiting aperture....
References
1. Vonrhein, C. and Schulz, G. E. 1999 Locating proper non-crystallographic symmetry in low-resolution electron-density maps with the program GETAX. Acta Cryst. D55, 225-229. 2. Matthews, B. W. 1968 Solvent content of protein crystals. J. Mol. Biol. 33,491-497. 3. Dodson, E. J., Winn, M., and Ralph, A. 1997 Collaborative computational project, number 4 providing programs for protein crystallography. Methods Enzymol. 277, 620-633. 4. Kantardjieff, K. A. and Rupp, B. 2003 Matthews coefficient...
Electron Density Maps
A model and two or more electron density maps are the basic inputs to the validation and model-building software, and both are usually obtained as output from a cycle of refinement see Chapter 13 . If the refinement program REFMAC 20 is used through the CCP4i interface 21 , it can be instructed to produce two aA-weighted maps 22 , one with coefficients 2mFo - DFc, acaic and another with coefficients mFo - DFc, acalc . The former shows positive density features for properly placed atoms that are...
References 1
1. Hoppe, W. 1957 Die 'Faltmolelciilmethode' eine neue Merhode zur Bestimmung der Kristallstruktur bei ganz oder teilweise bekannter Molekiilstruktur. Acta Crystallogr. 10, 750-751. 2. Rossmann, M. G. and Blow, D. M. 1962 The detection of sub-units within the crystallographic asymmetric unit. Acta Crystallogr. 15, 24-31. 3. Patterson, A. L. 1934 A fourier series method for the determination of the components of interatomic distances in crystals. Phys. Rev. 46, 372-376. 4. Crowther, R. A. and...
Data Processing
After diffraction data have been measured, one must reduce the data to extract observed structure factor amplitudes from the diffraction images. The steps involved are summarized in Fig. 16. Explicitly, for each diffraction spot in the images, one must assign it its general indices hkl , must measure the integrated intensity of that spot, must estimate the error of that measurement, and must make all known geometric corrections to that intensity, such as the Lorentz and polarization factors. In...
Summary of Programs Available
ACORN http www.ccp4.ac.uk . Primarily designed for solving protein structures at atomic resolution 1.2 A or better , but tests of substructure determinations were also reported 42 . Starts with a known substructure, known fragments positioned with an extensive molecular replacement search, or randomly placed atoms. This is followed by application of Patterson superposition methods, Sayre phase refinement, and a special density modification procedure dynamic density modification . CNS...
Sylvie Doubli
Macromolecular Crystallography Protocols M E T H O D S I N M O L E C U L A R B I O L O G Y 383. Cancer Genomics and Proteomics Methods and Protocols, edited by Paul B. Fisher, 2007 382. Microarrays, Second Edition Volume 2, Applications and Data Analysis, edited by Jang B. Rampal, 2007 381. Microarrays, Second Edition Volume 1, Synthesis Methods, edited by Jang B. Rampal, 2007 380. Immunological Tolerance Methods and Protocols, edited by Paul J. Fairchild, 2007 379. Glycovirology Protocols,...
Michael R Sawaya
Methods are presented for characterizing a crystal given an initial X-ray diffraction dataset. These methods can facilitate the structure determination process and illuminate the oligomeric state and symmetry of your molecule before the crystal structure is determined. Specifically, these methods include 1 calculation of Matthews coefficient to estimate the number of molecules in the asymmetric unit 2 calculation and interpretation of a self-rotation function to evaluate the point group...
Assembly of the Mounted Crystal Onto the XRay Diffraction Camera
1. Place the capillary-mounted crystal onto the goniometer head. The capillary can be affixed to the goniometer head using plasticine or through a brass-mounting pin if the crystal was mounted in a capillary with a pin. 3.3.2. Fiber Loop Mounts Propane as Cryogen 1. Adjust the position of the cold stream to provide more working space. The edge of the cold stream should be positioned approx 1-1.5 cm from the presumptive position of the crystal. 2. Select a vial containing a shock-cooled crystal...
TLS B Factor Parameterization
Probably the most significant, inappropriate constraint applied generally to protein models is the isotropic B factor. It is quite certain that atoms in crystals that diffract to resolutions lower than 2 A move anisotropically, and yet they are routinely modeled as isotropic. Although the excuse for this choice is the un deniable need to reduce the number of parameters in the model, this clearly is not a choice likely to improve the fit of the model to the data. Schomaker and Trueblood 11...
Detection of Merohedral Twinning by Examination of Intensity Distribution
Merohedral twinning can be diagnosed from a myriad of different symptoms. Some have been previously discussed, e.g., inability to interpret difference Patterson maps, observance of ghost peaks in Fourier maps, maintenance of a high Rfree after prolonged refinement, and molecular replacement solutions that overlap. But, these symptoms develop too late in the structure determination process and might be symptomatic of problems other than twinning. Earlier observations can aid identification of...
Notes Ogw
1. When adding a cryoprotectant, with or without extra compounds or derivatizing agents, to a hanging drop in situ, osmotic shock to the crystal can be reduced by placing the cryoprotectant solution next to but not yet joined with the drop bearing the crystal on the underside of the cover slip. After overnight equilibration by vapor diffusion, the cryoprotectant-bearing drop is dragged to the crystal drop, the cover slip is placed back over the reservoir, and the crystal is allowed to further...
HeavyAtom Detection
Several powerful programs for heavy-atom substructure detection are available like SHELXD 12 , Shake-and-Bake SnB 13 , HySS 14 , SOLVE 15 , or CRUNCH2 16 the procedures for heavy-atom substructure solution currently implemented in autoSHARP use either SHELXD or RANTAN 17 . However, solutions from other programs can be input through a simple file, in either Protein Data Bank PDB 18 or in our internal fractional coordinates format. The heavy-atom substructure for a given dataset or a collection...
Stereochemical Restraints
When a diffraction dataset is missing high-resolution reflections, the details of the molecule cannot be visualized. Fortunately, the molecule can be viewed as a set of bond lengths, bond angles, and torsion angles, instead of the conventional view of a set of atoms floating in space see Fig. 1 . The advantage derived from this geometrical view of a structure is that the values of the bond lengths and angles, and their standard uncertainties, can be predicted using high-resolution, small...
Processing Isomorphous Data
Whether one is using isomorphous methods to phase a new structure, or to study mutants or ligand-binding properties of an already-solved protein, the initial processing is very similar. One of the strengths of isomorphous methods is the speed with which they deliver useful answers. Within minutes of collecting and reducing isomorphous datasets, one can know whether one has a new derivative, Fig. 1. Contrast enhancing and phasing agents constellation of a mixture of iodine-containing compounds...
References Dcu
1. Branden, C. I. and Jones, T. A. 1990 Between objectivity and subjectivity. Nature 343, 687-689. 2. Kleywegt, G. J. and Jones, T. A. 1995 Where freedom is given, liberties are taken. Structure 3, 535-540. 3. Kleywegt, G. J. 2000 Validation of protein crystal structures. Acta Crystallogr. D56, 249-265. 4. Davis, A. M., Teague, S. J., and Kleywegt, G. J. 2003 Application and limitations of X-ray crystallographic data in structure-based ligand and drug design. Angew. Chem. Int. Ed. 42,...
Ccp4 Sad Insulin Bovine Crank Phasing
Fig. 3. Main autoSHARP output file for SIR AS experiments. 1. A refined heavy-atom model, comprising 1 position, occupancy, and temperature factor of the heavy-atom sites, 2 scaling parameters including anisotropic temperature factors , and 3 nonisomorphism parameters. 2. LLG or residual maps, which can be further analyzed to fine-tune the refinement parameters, e.g., typically to introduce anisotropic thermal parameters for certain sites. 3. Electron density maps from heavy-atom phasing,...
Miroslawa Dauter and Zbigniew Dauter
A short soak of protein crystals in cryosolution containing bromides or iodides leads to incorporation of these ions into the ordered solvent shell around the protein surface. The halide ions display significant anomalous signal, bromides in the vicinity of the absorption edge at 0.92 , and iodides at longer wavelengths, e.g., provided by the copper sources. Bromides can, therefore, be used through multiwavelength anomalous diffraction or single-wavelength anomalous diffraction SAD techniques...
Analysis of LLG Maps
Once convergence has been reached during the refinement of a given heavy-atom model, SHARP produces maps for the calculated heavy-atom density and for the LLG corresponding to each dataset. The final heavy-atom parameters and the associated maps are then analyzed in order to 1. Detect heavy-atom sites for which the refined parameters indicate a nonexistent site i.e., very low occupancy, very large temperature factor, or no significant density in the calculated model map these are deleted from...
Goniometer Head
beam. The exact location for the crystal in the capillary will depend on local geometry and should be verified prior to beginning the mounting procedure. 7. Using a paper wick of appropriate size, remove the mother liquor from both sides of crystal. Care should be exercised not to mechanically stress the crystal in any way. A small amount of mother liquor should be left on the syringe side of the crystal to prevent desiccation. Fig. 3. Mounting a macromolecular crystal into a capillary from the...
References Egl
1. Hendrickson, W. A. 1991 Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51-58. 2. Blundell, T. L. and Johnson, L. N. 1976 Protein Crystallography, Academic Press, London, UK. 3. McPherson, A. J. 1982 The Preparation and Analysis of Protein Crystals, Wiley, New York, NY. 4. Petsko, G. A. 1985 Preparation of isomorphous heavy-atom derivatives. Meth. Enzym. 114, 147-156. 5. Carvin, D., Islam, S. A., Sternberg, M. J. E., and Blundell,...
Info Ppl
aThe CNS routines are found in the inputs subdirectory of a standard installation. Major and minor menu headings are given for the CCP4i graphical user interface. aThe CNS routines are found in the inputs subdirectory of a standard installation. Major and minor menu headings are given for the CCP4i graphical user interface. 6. Generate phases by taking the inverse Fourier transform i.e., back-transform of an isomorphous model. In most cases the model PDB file to be used for generating phases is...
Soaking Procedure
The soaking procedure is very simple and consists of submerging the native crystal for a short time in the mother liquor containing simultaneously both a cryoprotectant and an appropriate halide salt, before freezing it for diffraction data collection. The halide ions diffuse into protein crystals very quickly. Experiments with variable soaking times have shown that soaking times as short as 10-15 s are sufficient. Longer soaking times may degrade the crystal diffraction power, but sometimes...
The Native Patterson Map Reveals the Presence of Pure Translational Symmetry
The native Patterson map reveals the presence of pure translational symmetry elements in a crystal. Recall that the native Patterson map contains peaks corresponding to interatomic vectors 8 . The Patterson function is given by the following equation. P u,v,w 1 v SZX Fhkl2 e-2ni hu kv lw h k l If one were to draw all possible vectors between atoms in one unit cell, then translate those vectors so that their tails lie at the origin, the heads of the vectors would correspond to the position of...