Ref: Crystallgraphy made crystal clear, Chapter 6. Preparation of heavy atom derivatives. Heavy-atom data bank:

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1 Obtaining Phases 1. Phasing by Isomorphous Replacement Methods SIR, Single Isomorphous Replacement method MIR, Multiple Isomorphous Replacement method (MIR) SIRAS, Single Isomorphous Replacement Anomalous Scattering 2. Phasing by MAD X-ray precession photographs of native and heavy atom derivative crystals. (a) Native lysozyme (hk0 zone); (b) lysozyme with p-chloromercuribenzene sulphonate. Ref: Crystallgraphy made crystal clear, Chapter 6. Preparation of heavy atom derivatives Heavy atom derivatization is most typically performed by either adding a heavy atom solution to the drops containing the crystal(s) (mother liquor) or transferring the crystal(s) from the mother liquor drop (where the crystal was grown) to a stabilizing solution containing the heavy atom. Possible concentrations of heavy atoms range from 1 mm to 100 mm depending upon the ph, temperature, crystallization reagent, and heavy atom. Heavy-atom data bank: Experimental Conditions for crystallization Chemical details of the heavy-atom compounds used Bibliographic References Atomic coordinates of heavy-atoms Details of binding sites of heavy-atoms/protein crystal environment Atomic coordinates of heavy-atoms/protein crystal environment 1

2 Isomorphism Derivative crystals usually display decreased diffraction due to radical induced radiation damage. They may show reduced lifetime in the beam, increased mosaicity to the point of disorder, cracking and dissolving. It is often necessary to determine a compromise between the soaking parameters (especially soaking time and derivative concentration) and level of substitution together with acceptable diffraction quality (say 3Å). This compromise can initially be determined by soaking, microscopic examination and taking X-ray exposures. The crystals must not display non-isomorphism. The unit cell parameters should be unchanged. Autoindexing will be required after collecting some data. Cell dimension changes of 0.5% will give intensity changes of 15% at 3Å. Cell dimension changes should be less than 1%. Isomorphous differences should decrease smoothly with increasing resolution. The average isomorphous difference ( F average ) is usually ~10 to 30%. If differences are due to non-isomorphism they will increase at higher resolution. It will be essential not to overestimate the usable resolution, which will not necessarily be limited by the resolution range of the data set, but often by lack of isomorphism at higher resolution. 2

3 Use Patterson function to locate heavy sites Real space Patterson maps Peaks in the Patterson function occur at endpoints of vectors between all pairs of atom sites, and therefore correspond to interatomic vectors. Given two atoms in the unit cell with coordinates x 1, y 1, z 1 and x 2, y 2, z 2 the Patterson will show a peak at the point uvw according to the relations u = x 1 -x 2 v = y 1 -y 2 w = z 1 -z 2 but also a peak at u = x 2 -x 1 v = y 2 -y 1 w = z 2 -z 1 Since both peaks represent vectors of equal size but of opposite direction, the Patterson will display additional inversion symmetry. The example shows four unit cells of space group P1 with 3 atoms per cell (blue) and four unit cells of Patterson space group P-1 with 6 peaks per cell + the origin (red). The unit cell in real space and Patterson space are identical. If the unit cell contains N atoms, the Patterson will show N 2 N peaks. The Patterson map will therefore be very crowded with many peaks overlapping. This is aggravated by Patterson peaks being less sharp than Fourier peaks. The combination of these two properties make the native Patterson of a macromolecule noninterpretable. However, assuming the derivative F PH and native F P amplitudes have been properly scaled, the Patterson map calculated from the isomorphous difference F PH - F P amplitudes is largely empty, so that it becomes possible to analyze and solve the Patterson. The difference Patterson function solely for heavy atoms in derivative crystals: 3

4 Harker Sections Example: Space group P2 1 equivalents positions: (x, y, z) and (-x, y+ ½, -z) (u, v, w) = (x, y, z) (-x, y+½, -z) = (2x, ½., 2z) = (-x, y+½, -z) (x, y, z) = (-2x, ½, -2z) Harker section is located at v = ½ (0.3, 0.5, 0.3) (0.7, 0.5, 0.7) (x, z) = (0.15, 0.15) Once we know where the heavy atoms are we can calculate their structure factors (F H ) : F H (h )= f H, j (h) exp (2πi hx j ) Obtaining phases from heavy-atom data Acentric case F P : Structure factor of native protein F P known, α P unknown F H : Structure factor of heavy atoms F H known, α H known F HP : Structure factor of heavy-atom derivative F PH known, α PH unknown In the case of ideal isomorphism: F HP = F H + F P F P = F HP -F H 4

5 Centric case The structure will always appear centrosymmetric when projected down an evenfold axis. So for example P2 1 (unique b) has one centric zone: h0l; P has three: h0l, hk0, 0kl. Space groups P1 and R3 have none. The centric zone reflections give a ready advantage in MIR studies, because α P can take only 1 of 2 possible values differing by 180 (eg 0/180 or 90/270 ). When α P = α H F PH (calc) = F P + F H. When α P = α H F PH (calc) = - F P + F H For each possibility of α P, F PH (calc) is compared with F PH (obs). Single Isomorphous Replacement (SIR) From our the experiment, we know only the magnitudes F HP (derivative) and Fp (protein) which can be represented in the complex plane as a circle of radius F HP and Fp, respectively. If we know both the magnitude and the phase of F H we can draw both circles offset by vector F H and obtain 2 solutions for possible phase values for Fp F P = F HP -F H At this point is is clear that the best phase we can obtain from the 2 solutions is the mean in between the 2 possibilities, and the phase error can be quite large. 5

6 Multiple Isomorphous Replacement (MIR) In order to eliminate the phase ambiguity we can prepare a second derivative and repeat the procedure. Provided the heavy atom is not at the same position, we can now obtain a unique solution for α (p), the phase of Fp : The theory is based on 2 assumptions : a) ideal isomorphism and b) exact heavy atom positions, neither of which are perfectly met, for practical and experimental reasons in the first case and for theoretical reasons in the second. In our picture it means that the phasing circles may not intersect in exactly in one spot, and another derivative may be necessary to improve the quality of the phases. The method is therefore called MIR, Multiple Isomorphous Replacement. The Importance of Phase This movie displays the effect of calculating a map with "wrong" phases. The "figure of merit" (cosine of the error in the phase) is displayed as "m". The images in this movie were calculated by mergeing a perfect calculated map with another map, calculated with the same amplitudes, but with phases obtained from a model with randomly positioned atoms. Mergeing these two maps always preserves the amplitudes, but changes the phases slowly to a new set of values. At what point do you think the map becomes untracable? The resolution here is 1.5A, and the R-factor is always 0.0%. 6

7 Multiple Anomalous Dispersion Phasing (MAD) In the vicinity of the X-ray absorption edge(s) of an element the wavelength dependent contributions f' and f" to the atomic X-ray scattering factor change rapidly with wavelength. This effect can be exploited to obtain exceptional initial phases for a structure. Regions of exceptional experimental electron density maps created from multiwavelength data collected at the ALS or SSRL. Ref: Crystallography made crystal clear, Chapter 6. What is anomalous scattering? When the incident photon has relatively low energy: The photon is either scattered or not, but is not absorbed as it has insufficient energy to excite any of the available electronic transitions. The photon scatters with no phase delay (imaginary, or f", component is 0). When the incident photon has high enough energy: Some photons are scattered normally. Some photons are absorbed and re-emitted at lower energy (fluorescence). Some photons are absorbed and immediately reemitted at the same energy (strong coupling to absorption edge energy). The scattered photon gains an imaginary component to its phase (f" scattering coefficient becomes non-zero); i.e. it is retarded compared to a normally scattered photon. 7

8 Scattering factors and anomalous scattering The scattering factor contains additional (complex) contributions from anomalous dispersion effects (essentially resonance absorption) which become substantial in the vicinity of the X-ray absorption edge of the scattering atom. f is real scattering component f is the imaginary component of the anomalous scattering. This effect is most easily measured as a function of x-ray energy by noting either the sharp increase in absorption or in fluorescence (see figure below). The imaginary scattering component f" is proportional to these directly measurable quantities. The real scattering component f' is related to f" via the Kramers- Kronig relationship. The Kramers-Kronig Equation The two components of the anomalous scattering factor, f" and f', are related by the Kramers-Kronig relationship. Once the f" spectrum is obtained experimentally from the sample crystal via fluorescence measurements, the corresponding f' spectrum may be calculated by numerical integration using the above equation. 8

9 X-ray Absorption Edges Here the interaction of the scattering atom with its chemical neighbors complicates the scattering behaviour considerably. Shown below is a comparison of the theoretical values of f' and f" with the values determined experimentally for the Cu site in single crystal of a blue copper protein [Guss et al, 1989]. In both cases the f' values were derived from the corresponding f" spectra via the Kramers-Kronig equation. 9

10 Estimate of signal from MAD experiment 1 Se atoms per 200 protein residues and 0 nucleic acid residues Se scattering factor estimates: f min = -9 f max = -2 f max = 6 Pessimistic scenario: 60% of anomalous scatters ordered; 60% of optimal f and f achieved If you want to detect a 3% signal then you would like R merge < It may look promising that even when your signal is only 3% at ~10Å. it rises to 5% at 2.5Å resolution. But remember that also means you need R merge < 5% in that 2.5Å shell! Rules of thumb: At least one Se for 17 kd 10

11 Choosing wavelengths for MAD data collection (inflection) (remote) (peak) - The largest signal will come from choosing the wavelength with maximal f'' ( 1 in the figure above). - The second wavelength is usually chosen to have maximal f' ( 2 in the figure above). - Additional wavelengths ( 3 and 4) are chosen at points remote from the absorption edge. Typically 3 and 4 are between 100eV and 1000eV from the absorption edge. Note: X-ray energy in kev = / in Å For a single type of anomalous scattering atom, i.e. a MAD experiment at a single absorption edge, you need a minimum of 2 wavelengths. It is better to be have more data points so that the set of simultaneous equations for MAD phasing is over-determined, so 3 wavelengths are better than 2. Beyond that it is a question of diminishing returns; 4 is better than 3, but may not be worth the extra data collection time for the slight improvement. MAD Phasing Phase diagram for MAD phasing ϕ T ϕ A = total observed scattering amplitude from our diffraction measurement at wavelength, which has an unknown phase we would dearly like to know F T = Normal scattering component of all atoms F A = Anomalous scattering contribution from all atoms = Difference in phase angle between normal and anomalous scattering components ϕ = ϕ T - ϕ A The basic idea is that if we can locate the anomalous scattering atoms within the unit cell (those contributing to F A ) then we can calculate the corresponding phase angle A. The MAD phasing equations of Karle [1980] can then be used to generate an estimate for and F T. In the simplest case we can then estimate the phase of the F T as + A. A Fourier transform of the amplitudes F T and phases ( + A ) should yield an electron density map corresponding to all atoms in the structure. 11

12 Unknown Each of our measurements of F(h) at some wavelength gives us one instance of the equation above, and the separate instances may be treated as a system of simultaneous equations from which we want to obtain the quantities F A, F T, and ϕ. Actually we have two measurements for each wavelength, because the observation for F - (h) may be treated as an observation of F + (h) with the value of f inverted. So to obtain values of our three unknowns we will need at least three observations, which is to say data collected at two wavelengths with distinct scattering factors. Obviously it is better to be over-determined, so we would prefer data collected at three or more wavelengths. Note: The treatment of data collected from crystals with more than one type of anomalous scattering atoms is exactly parallel, with the addition of two new quantities to be estimated for each new scattering type k: FAk and k. So for two anomalous scattering types we have 5 unknowns and we will need data from a minimum of three wavelengths (F+ and F- at each of 3 wavelengths = 6 observations to derive 5 unknowns). Locating the anomalous scatters by Patterson maps You've collected your MAD data and run it through a phasing program that has produced initial estimates for the three quantities F T, F A, and ϕ. How do you get from there to a phase estimate for the protein, ϕ T? ϕ T= ϕ A + ϕ So you need to locate the anomalous scattering atoms in the unit cell, so that you can calculate the phase ϕ A of their contribution to the total scattering. After you have ϕ T and F T, a MAD map (α hkl = ϕ T and F hkl = F T ) can be calculated: 12

13 Terrific MAD Maps MAD phases result from perfectly isomorphous data, and do not detoriate at low resolution as do MIR phases. MIR phases, on the other hand, provide superior phasing power at low resolution resulting in better connectivity. Not surprising, the best maps we every have seen were either MAD or combined MIR-MAD maps. Region of exceptional experimental electron density map created from multi-wavelength data collected at the ALS. Only solvent flattening and density modification have been performed. No phase combination with any model phases has taken place. Panel 2 :.to show the excellent correspondence with actual protein residues, a previously determined molecular replacement model of Arg has been placed into the electron density. The model can be unambiguously and easily built into the electron density. Panel 3 : View is a cross section of the apoe four helix bundle. The empty space in the center of helix one and two is clearly visible. Panel 4: : to show the excellent correspondence of experimental electron density with actual protein residues, model helices have been placed into the electron density. Experimental phases had an average figure of merit of There is very little noise and excellent connectivity in the electron density. MIR: Collect data at a single wavelength for native and heavy atom derivative crystals MAD: Collect data at different wavelengths from a single heavy-atom containing crystal 13

14 MIR: Scale heavy atom data F ph to native data F p MAD: Scale F λ1, F λ2, F λ2, etc. MIR: Calculate difference Patterson ( F p - F ph ) 2 MAD: Calculate anomalous difference Patterson ( F + - F - ) 2 14

15 Transcription- A template DNA strand is transcribed into a complementary RNA chain by RNA polymerase Transcription of DNA into RNA is catalyzed by RNA polymerase, which can initiate the synthesis of strands de novo on DNA templates. 1. The ribonucleotide to be added at the 3 end of a growing RNA strand is specified by base pairing between the next base in the template DNA strand and the incoming ribonucleotiside triphosphate (rntp). 2. The nucleotide at the 5 end of an RNA strand retains all three of its phosphate groups; 3. all subsequent nucleotides release pyrophosphate (PPi) when added to the chain and retain only their a phosphate (red). The released PPi is subsequently hydrolyzed by pyrophosphatase to Pi, driving the equilibrium of the overall reaction toward chain elongation. 4. In most cases, only one DNA strand is transcribed into RNA. +1: the site at which RNA polymerase starts transcription 15

16 Stages in Transcription 16

17 The yeast Saccharomyces cerevisiae RNA polymerase II comprises 12 different polypeptides with a total mass of about 0.5 megadaltons (MD). The crystal structure of yeast Pol II contains 10 subunits. Three major obstacles before phase determination 1. Diffraction to 3.5 Å resolution could not be obtained reproducibly. 2. The crystals were non-isomorphous, varying by as much as 10 Å in one dimension of the unit cell. Very few crystals could be derivatized and matched with an isomorphous native crystal. 3. Heavy atom compounds commonly used for protein phase determination destroyed diffraction from the crystals. These difficulties were overcome in the present work by a soaking procedure that shrank the crystals to an apparent minimum of the variable unit cell dimension. The resulting crystals were isomorphous and diffracted isotropically to 3.0 Å resolution. 17

18 Purification of yeast pol II and crystallization in the orthorhombic space group I222 were as described [L. Myers et al., Methods Companion Methods Enzymol. 12, 212 (1997); (29)]. The crystals, containing a single pol II in the asymmetric unit, were transferred under argon/hydrogen in seven steps from harvest buffer [390 mm (NH 4 ) 2 HPO 4 /NaH 2 PO 4, ph 6.0, 16% PEG 6000, 50 mm dioxane, and 3 mm dithiothreitol (DTT)] to stabilization buffer (100 mm MES, ph 6.3, 16% PEG 6000, 350 mm NaCl, 17% PEG 400, 50 mm dioxane, and 3 mm DTT). Crystals were cooled to 4 o C overnight and maintained at that temperature for 5 days before flash-cooling. This treatment caused shrinkage along the crystallographic a axis to 131 Å, extended the diffraction limit, and led to high isomorphism of the crystals. Crystals were mounted at 4 o C in nylon loops 200 mm in diameter, plunged into liquid nitrogen and stored for data collection. Because the improved crystals were non-isomorphous with the original crystals, initial phases were redetermined by multiple anomalous dispersion (MAD) with a six tantalumatom cluster derivative, which showed a single peak in difference Pattersons. Localization of heavy atoms. Harker sections of isomorphous and anomalous difference Patterson maps of the tantalum cluster derivative. A single peak at the same position in the two maps is observed. Heights of the Harker peaks in the isomorphous and anomalous difference Pattersons were 6 σ and 5 σ, respectively. The resolution range of the data used is 40 to 5.5 Å. The contour levels are 3 σ (background) and 1 σ (steps). 18

19 These phases sufficed to reveal individual heavyatoms in other crystals by means of cross-difference Fouriers. F hkl = F = ( F + - F - ) Native data collected at the zinc absorption edge. Therefore only zinc peaks can be seen in the maps. MAD phases: calculated using Tantalum MAD data MIRAS phases: calculated from nine derivative. Anomalous difference Fourier calculated with native data collected at the zinc anomalous peak energy using initial tantalum MAD phases (left) and final MIRAS phases (right). The projection of one asymmetric unit along the z axis is shown for tantalum and MIRAS phases at a contour level of 3 s and 7 s, respectively, with 1 s steps. The eight strong peaks correspond to structural zinc atoms. The ninth peak corresponds to the active site metal and likely arises from partial replacement of magnesium by zinc. Phases were determined by multiple isomorphous replacement with anomalous scattering (MIRAS) from 10 data sets, ranging from 4.0 to 3.1 Å resolution. 19

20 Yeast RNA Polymerase Structure Yeast RNA Polymerase II at 2.8 Å Resolution The clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. Two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. Ref: Science, 2001, 292, Science, 2000, 288,

21 Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Angstrom Resolution (Science 2001, 292, ) Comparison of structures of free pol II (top) and the pol II transcribing complex (bottom). The clamp (yellow) closes on DNA and RNA, which are bound in the cleft above the active center. The remainder of the protein is in gray. Structure of the pol II transcribing complex. The template DNA strand is in blue, nontemplate strand in green, and RNA in orange. The Rpb1 bridge helix traversing the cleft is highlighted in green. The active site metal A is shown as a pink sphere. Nucleic acids in the transcribing complex and their interactions with pol II. (A) (B) The main technical challenge of this work was the isolation and crystallization of a transcribing complex. Initiation at an RNA polymerase II promoter requires a complex set of general transcription factors and is poorly efficient in reconstituted systems. Moreover, most preparations contain many inactive polymerases, and the transcribing complexes obtained would have to be purified by mild methods to preserve their integrity. The initiation problem was overcome with the use of a DNA duplex bearing a single-stranded tail at one 3 -end. Pol II starts transcription in the tail, two to three nucleotides from the junction with duplex DNA, with no requirement for general transcription factors. All active polymerase molecules are converted to transcribing complexes, which pause at a specific site when one of the four nucleoside triphosphates is withheld. The problem of contamination by inactive polymerases was solved by passage through a heparin column; inactive molecules were adsorbed, whereas transcribing complexes flowed through, presumably because heparin binds in the positively charged cleft of the enzyme, which is occupied by DNA and RNA in transcribing complexes. DNA (tailed template) and RNA sequences. DNA template and non-template strands are in blue and green, respectively, and RNA is in red. Ordering of nucleic acids in the transcribing complex structure. Nucleotides in the solid box are well ordered. Nucleotides in the dashed box are partially ordered, whereas those outside the boxes are disordered. Three protein regions that abut the downstream DNA are indicated. 21

22 Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3 end of the RNA in the nucleotide addition site. The 3 end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5 -most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of switches at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein- nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, abortive cycling during transcription initiation, and RNA and DNA translocation during transcription elongation. Molecular replacement Determination of initial phases for an unknown structure by positioning a molecule (or fragment of it) in the cell of the unknown. Model Building and phase improvement Structure refinement Crystallographic refinement is the modification of an atomic model to improve the agreement between observed and calculated structure factors (or intensities) while maintaining reasonable chemical restraints. Structure validation PDB Reading: Crystallography made crystal clear, Chapters 7 and 8. References:

23 Phases may be calculated given a known structure. For proteins there are just too many atoms to guess all their individual positions. However, if you already know the positions of atoms within one molecule then instead of placing atoms in the unit cell, you "only" have to place molecules in the unit cell [Molecular (Re)placement; here abbreviated as MR]. One way to solve the phase problem is to possess an atomic model, from which estimates of the phases can be computed. The prior knowledge of the protein structure, the "search model", can come from several sources: -Same protein solved in different space group -Mutant or complex of known native protein -Homologous protein -NMR/theoretical models -Fragments (domains) of multiple proteins As a rule of thumb, molecular replacement will probably be fairly straightforward if the model is fairly complete and shares at least 40% sequence identity with the unknown structure. It becomes progressively more difficult as the model becomes less complete or shares less sequence identity. 23

24 Molecular Replacement Stages Target 1. Rotation Search Ω Search model 3. Translation Search 2. PC Refinement 4. PC Refinement 5.Rigid Body Refinement 24

25 Patterson map P(uvw) = (1 / V) Σ h Σ k Σ l F (hkl) 2 exp[ -2πi(hu + kv + lw)] The Patterson map is basically a Fourier map calculated with the square of the structure factor amplitude and a phase of zero. Patterson showed that this type of map is basically an interatomic vector map. Each peak in the map corresponds to a vector between atoms in the crystal and the intensity of the peak is the product of the electron densities of each atom. Patterson map is a vector map, with peaks at the positions of vectors between atoms in the unit cell. Intramolecular vectors (from one atom in the molecule to another atom in the same molecule) depend only on the orientation of the molecule, and not on its position in the cell, so these can be exploited in the rotation function. Intermolecular vectors depend both on the orientation of the molecule and on its position so, once the orientation is known, these can be exploited in the translation function. Cross-rotation search to determine rotation 25

26 Rotation Function To quantify the agreement between search and target Pattersons the following function was proposed by Rossmann and Blow (1962). In order to quantify the overlap between the observed and calculated Pattersons we use a product function. In this procedure, the corresponding positions in the observed and calculated Patterson maps are multiplied. This gives a maximum when the two Pattersons overlap. 26

27 R-factor and correlation-coefficient translation functions 2 27

28 Structure Refinement Refinement is the process of adjusting the model to find a closer agreement between the calculated and observed structure factors. Least squares refinement Given these data, what are the parameters of the model that give the minimum variance of the observations? Stereochemically restrained least-squares refinement To use known molecular geometry to reduce the number of variables or to treat the additional information as additional observations. X-ray restrain Restrains the distance between atoms Restrains the planarity of aromatic rings Restrains the configuration to the correct enantiomer Introduce restrains for nonbonded or van der Waals contacts Restrains torsion angles 28

29 Estimating the accuracy of the structural model 1. Crystallographic R-factors Brunger introduced the free R-factor, which is unbiased by the refinement process. In this method one divides the reflections into a test set (T) of unique reflections and a working set (W). The refinement is carried out with the working set only, and the free-r-factor is calculated with the test set of reflections only. The Rfree is commonly 2-8% higher than the regular R-factor. 29

30 2. Ramachandran plot from PROCHECK Is the geometry "tight"? Bond angles within 3 and bond lengths within 0.03 Å of expected values; fewer than 5% of non-glycine residues are Ramachandran outliers. 3. Luzzati method Luzzati has observed a relationship between the average error r and the difference between Fobs and Fcalc. Therefore, a curve of R-factor vs. sinθ/λ can be compared with a family of calculated lines. From the line that is closest to the experimental curve, the coordinate error for the crystal structure is derived Å 0.12 Å 0.08 Å 30

31 4. The 3D-1D Profile Method VERIFY3D Measure the compatibility of a protein model with its own amino acid sequence. ERRAT Analyze the relative frequencies of noncovalent interactions (CC, CN, CO, NN, NO, and OO) between atoms of various types. 31

32 ERRAT VERIFY Crystallographic Databases The Cambridge Structural Database (CSD) 32

33 Protein Data Bank (PDB) Research Collaboration for Structural Bioinformatics As of Tuesday Dec 05, 2006, there are Structures Molecule Type Proteins Nucleic Acids Protein/NA Complexes Other Total X-ray NMR Exp. Method Electron Microscopy Other Total PDB Content Growth Bovine heart cytochrome c oxidase (422 kd), calcium pump of sarcoplasmic reticulum (110 kd), bacterial RNA polymerase holoenzyme (400 kd), bacterial ribosome (2,500 kd), yeast ribosome (). 33

34 Reference: Present at the Flood- How Structural Molecular Biology Came About, by Richard E. Dickerson, Published by Sinauer Associates, Inc, Dickerson s law n = exp [0.19 (Year-1960)] Predicted Solved protein crystal structures Year ,066 12,123 Year ,342 ~37,107 Year 2033 > one million per year >3000 per day 34

35 HEADER HYDROLASE 12-JUN-02 1M08 TITLE CRYSTAL STRUCTURE OF THE UNBOUND NUCLEASE DOMAIN OF COLE7 COMPND MOL_ID: 1; COMPND 2 MOLECULE: COLICIN E7; COMPND 3 CHAIN: A, B; COMPND 4 FRAGMENT: NUCLEASE DOMAIN; COMPND 5 EC: ; COMPND 6 ENGINEERED: YES SOURCE MOL_ID: 1; SOURCE 2 ORGANISM_SCIENTIFIC: ESCHERICHIA COLI; SOURCE 3 ORGANISM_COMMON: BACTERIA; SOURCE 4 STRAIN: W3110; SOURCE 5 GENE: COLE7 OR CEA; SOURCE 6 EXPRESSION_SYSTEM: ESCHERICHIA COLI; SOURCE 7 EXPRESSION_SYSTEM_COMMON: BACTERIA; SOURCE 8 EXPRESSION_SYSTEM_STRAIN: M15; SOURCE 9 EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID; SOURCE 10 EXPRESSION_SYSTEM_PLASMID: PQE70 KEYWDS HNH MOTIF, ENDONUCLEASE, COLICIN, ZN-BINDING PROTEIN EXPDTA X-RAY DIFFRACTION AUTHOR Y.S.CHENG,K.C.HSIA,L.G.DOUDEVA,K.F.CHAK,H.S.YUAN JRNL AUTH Y.S.CHENG,K.C.HSIA,L.G.DOUDEVA,K.F.CHAK,H.S.YUAN JRNL TITL THE CRYSTAL STRUCTURE OF THE NUCLEASE DOMAIN OF JRNL TITL 2 COLICIN E7 SUGGESTS A MECHANISM FOR BINDING TO JRNL TITL 3 DOUBLE-STRANDED DNA BY THE HNH ENDONUCLEASES JRNL REF TO BE PUBLISHED JRNL REFN REMARK 1 REMARK 1 REFERENCE 1 REMARK 1 AUTH T.P.KO,C.C.LIAO,W.Y.KU,K.F.CHAK,H.S.YUAN REMARK 1 TITL THE CRYSTAL STRUCTURE OF THE DNASE DOMAIN OF REMARK 1 TITL 2 COLICIN E7 IN COMPLEX WITH ITS INHIBITOR IM7 REMARK 1 TITL 3 PROTEIN REMARK 1 REF STRUCTURE V REMARK 1 REFN ASTM STRUE6 UK ISSN

36 REMARK 2 REMARK 2 RESOLUTION ANGSTROMS. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : CNS 1.0 REMARK 3 AUTHORS : BRUNGER,ADAMS,CLORE,DELANO,GROS,GROSSE- REMARK 3 : KUNSTLEVE,JIANG,KUSZEWSKI,NILGES, PANNU, REMARK 3 : READ,RICE,SIMONSON,WARREN REMARK 3 REMARK 3 REFINEMENT TARGET : ENGH & HUBER REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 2.10 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : REMARK 3 DATA CUTOFF (SIGMA(F)) : REMARK 3 OUTLIER CUTOFF HIGH (RMS(ABS(F))) : NULL REMARK 3 COMPLETENESS (WORKING+TEST) (%) : 93.6 REMARK 3 NUMBER OF REFLECTIONS : REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING SET) : REMARK 3 FREE R VALUE : REMARK 3 FREE R VALUE TEST SET SIZE (%) : REMARK 3 FREE R VALUE TEST SET COUNT : 1603 REMARK 3 ESTIMATED ERROR OF FREE R VALUE : REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM LUZZATI PLOT (A) : 0.22 REMARK 3 ESD FROM SIGMAA (A) : 0.13 REMARK 3 LOW RESOLUTION CUTOFF (A) : 5.00 REMARK 3 REMARK 3 CROSS-VALIDATED ESTIMATED COORDINATE ERROR. REMARK 3 ESD FROM C-V LUZZATI PLOT (A) : 0.29 REMARK 3 ESD FROM C-V SIGMAA (A) : 0.20 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A) : REMARK 3 BOND ANGLES (DEGREES) : 1.50 REMARK 3 DIHEDRAL ANGLES (DEGREES) : REMARK 3 IMPROPER ANGLES (DEGREES) : 0.93 REMARK 3 REMARK 3 ISOTROPIC THERMAL MODEL : RESTRAINED REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. RMS SIGMA REMARK 3 MAIN-CHAIN BOND (A**2) : ; REMARK 3 MAIN-CHAIN ANGLE (A**2) : ; REMARK 3 SIDE-CHAIN BOND (A**2) : ; REMARK 3 SIDE-CHAIN ANGLE (A**2) : ;

37 REMARK 100 REMARK 100 THIS ENTRY HAS BEEN PROCESSED BY RCSB ON 17-JUN REMARK 100 THE RCSB ID CODE IS RCSB REMARK 200 REMARK 200 EXPERIMENTAL DETAILS REMARK 200 EXPERIMENT TYPE : X-RAY DIFFRACTION REMARK 200 DATE OF DATA COLLECTION : 08-AUG-2001 REMARK 200 TEMPERATURE (KELVIN) : REMARK 200 PH : 7.00 REMARK 200 NUMBER OF CRYSTALS USED : 1 REMARK 200 REMARK 200 SYNCHROTRON (Y/N) : N REMARK 200 RADIATION SOURCE : ROTATING ANODE REMARK 200 BEAMLINE : NULL REMARK 200 X-RAY GENERATOR MODEL : RIGAKU RU300 REMARK 200 MONOCHROMATIC OR LAUE (M/L) : M REMARK 200 WAVELENGTH OR RANGE (A) : REMARK 200 MONOCHROMATOR : NULL REMARK 200 OPTICS : MIRRORS REMARK 200 REMARK 200 DETECTOR TYPE : IMAGE PLATE REMARK 200 DETECTOR MANUFACTURER : RIGAKU RAXIS II REMARK 200 INTENSITY-INTEGRATION SOFTWARE : DENZO REMARK 200 DATA SCALING SOFTWARE : SCALEPACK REMARK 200 REMARK 200 NUMBER OF UNIQUE REFLECTIONS : REMARK 200 RESOLUTION RANGE HIGH (A) : REMARK 200 RESOLUTION RANGE LOW (A) : REMARK 200 REJECTION CRITERIA (SIGMA(I)) : REMARK 200 REMARK 200 OVERALL. REMARK 200 COMPLETENESS FOR RANGE (%) : 95.0 REMARK 200 DATA REDUNDANCY : REMARK 200 R MERGE (I) : NULL REMARK 200 R SYM (I) : REMARK 200 <I/SIGMA(I)> FOR THE DATA SET : REMARK 200 REMARK 200 IN THE HIGHEST RESOLUTION SHELL. REMARK 200 HIGHEST RESOLUTION SHELL, RANGE HIGH (A) : 2.10 REMARK 200 HIGHEST RESOLUTION SHELL, RANGE LOW (A) : 2.18 REMARK 200 COMPLETENESS FOR SHELL (%) : 92.7 REMARK 200 DATA REDUNDANCY IN SHELL : NULL REMARK 200 R MERGE FOR SHELL (I) : NULL REMARK 200 R SYM FOR SHELL (I) : REMARK 200 <I/SIGMA(I)> FOR SHELL : REMARK 200 REMARK 200 DIFFRACTION PROTOCOL: SINGLE WAVELENGTH REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT REMARK 200 SOFTWARE USED: CNS REMARK 200 STARTING MODEL: 7CEI REMARK 200 REMARK 200 REMARK: NULL 37

38 REMARK 280 REMARK 280 CRYSTAL REMARK 280 SOLVENT CONTENT, VS (%): 48.5 REMARK 280 MATTHEWS COEFFICIENT, VM (ANGSTROMS**3/DA): 2.40 REMARK 280 REMARK 280 CRYSTALLIZATION CONDITIONS: SODIUM PHOSPHATE, SODIUM CHLORIDE, REMARK 280 ZINC CHLORIDE, AMMONIUM ACETATE REMARK 290 REMARK 290 CRYSTALLOGRAPHIC SYMMETRY REMARK 290 SYMMETRY OPERATORS FOR SPACE GROUP: P 1 REMARK 290 REMARK 290 SYMOP SYMMETRY REMARK 290 NNNMMM OPERATOR REMARK X,Y,Z REMARK 290 REMARK 290 WHERE NNN -> OPERATOR NUMBER REMARK 290 MMM -> TRANSLATION VECTOR REMARK 290 REMARK 290 CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS REMARK 290 THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM REMARK 290 RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY REMARK 290 RELATED MOLECULES. REMARK 290 SMTRY REMARK 290 SMTRY REMARK 290 SMTRY REMARK 290 REMARK 290 REMARK: NULL REMARK 300 REMARK 300 BIOMOLECULE: 1 REMARK 300 THIS ENTRY CONTAINS THE CRYSTALLOGRAPHIC ASYMMETRIC UNIT REMARK 300 WHICH CONSISTS OF 2 CHAIN(S). SEE REMARK 350 FOR REMARK 300 INFORMATION ON GENERATING THE BIOLOGICAL MOLECULE(S). REMARK 900 REMARK 900 RELATED ENTRIES REMARK 900 RELATED ID: 7CEI RELATED DB: PDB REMARK 900 7CEI CONTAINS THE COMPLEX STRUCTURE BETWEEN THE NUCLEASE REMARK 900 DOMAIN OF COLE7 AND ITS INHIBITOR IM7 PROTEIN REMARK 900 RELATED ID: 1CEI RELATED DB: PDB REMARK 900 1CEI CONTAINS THE CRYSTAL STRUCTURE OF THE IM7 PROTEIN DBREF 1M08 A SWS Q47112 CEA7_ECOLI DBREF 1M08 B SWS Q47112 CEA7_ECOLI SEQADV 1M08 MET A 446 SWS Q47112 INITIATING MET SEQADV 1M08 MET B 446 SWS Q47112 INITIATING MET SEQRES 1 A 131 MET ARG ASN LYS PRO GLY LYS ALA THR GLY LYS GLY LYS SEQRES 2 A 131 PRO VAL ASN ASN LYS TRP LEU ASN ASN ALA GLY LYS ASP SEQRES 3 A 131 LEU GLY SER PRO VAL PRO ASP ARG ILE ALA ASN LYS LEU SEQRES 4 A 131 ARG ASP LYS GLU PHE LYS SER PHE ASP ASP PHE ARG LYS SEQRES 5 A 131 LYS PHE TRP GLU GLU VAL SER LYS ASP PRO GLU LEU SER SEQRES 6 A 131 LYS GLN PHE SER ARG ASN ASN ASN ASP ARG MET LYS VAL SEQRES 7 A 131 GLY LYS ALA PRO LYS THR ARG THR GLN ASP VAL SER GLY SEQRES 8 A 131 LYS ARG THR SER PHE GLU LEU HIS HIS GLU LYS PRO ILE SEQRES 9 A 131 SER GLN ASN GLY GLY VAL TYR ASP MET ASP ASN ILE SER SEQRES 10 A 131 VAL VAL THR PRO LYS ARG HIS ILE ASP ILE HIS ARG GLY SEQRES 11 A 131 LYS SEQRES 1 B 131 MET ARG ASN LYS PRO GLY LYS ALA THR GLY LYS GLY LYS SEQRES 2 B 131 PRO VAL ASN ASN LYS TRP LEU ASN ASN ALA GLY LYS ASP SEQRES 3 B 131 LEU GLY SER PRO VAL PRO ASP ARG ILE ALA ASN LYS LEU SEQRES 4 B 131 ARG ASP LYS GLU PHE LYS SER PHE ASP ASP PHE ARG LYS SEQRES 5 B 131 LYS PHE TRP GLU GLU VAL SER LYS ASP PRO GLU LEU SER SEQRES 6 B 131 LYS GLN PHE SER ARG ASN ASN ASN ASP ARG MET LYS VAL SEQRES 7 B 131 GLY LYS ALA PRO LYS THR ARG THR GLN ASP VAL SER GLY SEQRES 8 B 131 LYS ARG THR SER PHE GLU LEU HIS HIS GLU LYS PRO ILE SEQRES 9 B 131 SER GLN ASN GLY GLY VAL TYR ASP MET ASP ASN ILE SER SEQRES 10 B 131 VAL VAL THR PRO LYS ARG HIS ILE ASP ILE HIS ARG GLY SEQRES 11 B 131 LYS HET ZN2 A HET PO4 A HET ZN2 B HET PO4 B

39 ATOM 1 N MET A N ATOM 2 CA MET A C ATOM 3 C MET A C ATOM 4 O MET A O ATOM 5 CB MET A C ATOM 6 CG MET A C ATOM 7 SD MET A S ATOM 8 CE MET A C ATOM 9 N ARG A N ATOM 10 CA ARG A C ATOM 11 C ARG A C ATOM 12 O ARG A O ATOM 13 CB ARG A C ATOM 14 CG ARG A C ATOM 15 CD ARG A C ATOM 16 NE ARG A N ATOM 17 CZ ARG A C ATOM 18 NH1 ARG A N ATOM 19 NH2 ARG A N ATOM 20 N ASN A N ATOM 21 CA ASN A C ATOM 22 C ASN A C ATOM 23 O ASN A O ATOM 24 CB ASN A C ATOM 25 CG ASN A C ATOM 26 OD1 ASN A O ATOM 27 ND2 ASN A N ATOM 28 N LYS A N ATOM 29 CA LYS A C ATOM 30 C LYS A C PyMol: 39