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Low Resolution Phasing
XENON Derivatives

PROTEIN CRYSTALLOGRAPHY
Very Low Resolution Data Collection on Stations 7.2 and 9.5

Rationale
Technical Issues for 7.2 | Protocol for 7.2 | Experiments on 7.2
Technical Issues for 9.5 | Protocol for 9.5 | Experiments on 9.5
References | Contact

Rationale

The low resolution reflections are the most important terms in determining the molecular envelope, or the shape, of a protein. This can be used to supplement phase information from elsewhere (e.g. from SIR or single wavelength anomalous scattering). It can also provide a check for models of the solvent contribution in structure refinement.

The low resolution terms, down to 250Å, are now easily accessible using a new camera, built at EMBL Hamburg, and installed on station 7.2 at the SRS. Low resolution data is also accessible on station 9.5. Information is given here for those who want to make use of this facility during their beamtime on either station.

One of the purposes of making this available is that it will assist in evaluating the usefulness of the low resolution data. Gerard Bricogne and his group have indicated that they would be interested in looking at data relevant to supplementing phases obtained from SIR or single wavelength studies. They are also interested in the use of such data as a check for modeling the solvent contribution. The group from Strasbourg and Puschino (Urzhumtsev and Lunin, see refs below) also have an interest in these areas. People with low resolution data might like to contact these groups directly to collaborate in investigating the use of the data.

Some references are given to the use of the low resolution data. Note that those in the CCP4 Study Weekend Proceedings can be accessed via:
http://www.dl.ac.uk/CCP/CCP4/proceedings/1997/details97.html

Technical Issues for 7.2

Low order reflections can of course be collected using a laboratory based source, provided care is taken to collimate the beam, reduce the scatter and align a small beamstop. However, the set up on Station 7.2 has the following features which make it easy to use.

  1. A synchrotron source so the flux is maintained even for a finely collimated beam.
  2. The standard wavelength of 1.488Å on Station 7.2 gives some advantage over 0.9Å for separating the low order spots from the direct beam.
  3. The Hamburg PX camera, which features a long collimator and beamstop translation.
Protocol for Collecting Very Low Resolution Data on Station 7.2.

  1. Collimate the beam to 150-100 microns using the slits on the Hamburg PX Camera
  2. Collimate the beam at the monochromator using the pre-mono slits.
  3. Move the beamstop away from the crystal by ~10cm.
  4. Move the MAR detector back along the optical bench (maximum distance is currently 916mm).
Daresbury staff require approximately one hour to prepare Station 7.2 for low resolution data collection.
Experiments on 7.2

Two experiments are described here. They both involved using the new EMBL camera on station 7.2. It features beam collimating slits and a moveable beamstop which facilitate the collection of very low resolution (vlr) data from protein crystals. The beam was slit down to 100 microns, the beamstop was moved approximately 10 cm out. The detector to crystal distance was set to 500 mm for mutant malto-porin, and 916 mm for human Ceruloplasmin. The latter distance is the largest accessible currently. If a shorter distance is used, some 'medium' resolution data might be accessible, say to 6Å, which would facilitate merging vlr data with the highest resolution that the sample affords. A reasonable number of reflections would then be available to help with sensible scaling.

A crystal of mutant malto-porin was provided by Tilman Schirmer, Biocentre, University of Basel. It is a pore forming protein, in the bacterial membrane. The wild-type structure showed a trimeric cluster of beta-barrels. The mutations affect the inside of the barrel, and therefore are not likely to interfere with lattice contacts. The cell dimensions are isomorphous with the wild-type. Data collection was completed to 3.0Å resolution, and then followed by the low resolution pass. The details of the vlr experiment are shown in the table below.

A crystal of human ceruloplasmin was provided by Slava Zaitsev from DL. Ceruloplasmin is a ferroxidase that mediates the release of iron in the blood, and its subsequent incorporation into serum transferrin. The crystal was mounted in a capillary at 4oC and maintained at this temperature once mounted on the camera by setting the cryocooler to 277 K. The data collection parameters and processing statistics are outlined in the table below.

Crystal Malto-porin hCP
SRS Station 7.2 7.2
Wavelength(Å) 1.488 1.488
Temperature(K) 293 277
Rotation Range (total) 120o 72o
Rotation Step 3o 2o
Resolution Limits(Å) 200 - 8.5 185 - 20
Spacegroup C2221 P3221
Unit Cell (Å) a=130.1,
b=213.0,
c=217.6
a=b=213.91,
c=85.00
Reflections Measured 10746 588
Unique Reflections 2709 124
Merging R-Factor (%) 3.5 4.8
Completeness (%) 92.2 72.8
Multiplicity 4.0 4.7

Table 1: Data collection parameters and processing statistics for the low resolution data collection of Malto-Porin and Human Ceruloplasmin. The data were processed using MOSFLM, and scaled with SCALA.

N.B. The completeness of the hCP data set was low due to shortage of time. The porin data set completeness could not be improved, although 120 deg. of rotation were covered. It must be assumed that the orientation of the crystal has an important bearing on the result. Users should aim for higher completeness where possible.

composite diffraction pattern typical diffraction pattern
A composite diffraction pattern covering the full rotation range, from the malto- porin crystal. The circles correspond to resolutions of 100, 133, 200 and 400 Å respectively (outer to inner). The (111) reflection was the lowest order measured. The higher resolution data extended to 8.35Å, but are not shown here. A typical diffraction pattern obtained during the low resolution data collection from human ceruloplasmin. The circles correspond to resolutions of 200, 100, 67 and 50Å respectively. The (100) reflection was the lowest order collected.
Technical Issues for 9.5

  1. A synchrotron source so the flux is maintained even for a finely collimated beam.
  2. The easily tunable wavelength (0.7-1.5 Å) allows flexibility in separating the low order spots and reducing air scatter. This also permits the possibility of Multi-wavelength Anomalous Solvent Contrast (MASC) experiments (see reference 11).
  3. The detector can be translated back along the optical bench up to 1400 mm.
  4. The beamstop can be positioned anywhere between the sample and the detector.
  5. Pre-collimator slits are available to reduce the beam size.
Protocol for Collecting Very Low Resolution Data on Station 9.5
  1. Set the monochromator to the rquired wavelength (0.7 to 1.5 Å).
  2. Collimate the beam to 100-200 microns using the pre-collimator slits.
  3. Move the beamstop away from the sample.
  4. Move the MAR detector back along the optical bench (maximum distance approx. 1400 mm).
  5. An optional He path is available to place between the beamstop and detector, in order to reduce the absorption of X-rays in air.
Experiments on 9.5

Under construction

Very low resolution experiments have been conducted by Gwyndaf Evans of the MRC-LMB Cambridge on station 9.5 of the SRS.

Low resolution diffraction pattern1

Low resolution diffraction pattern collected on station 9.5. The circles correspond to resolutions of 80, 40, 27 and 20 Å respectively.

Low resolution diffraction pattern2

References

  1. Urzhumtsev, A., Lunin, V. & Podjarny, A. (1997).
    Recent Advances in Phasing,
    Proceedings of the CCP4 Study Weekend, pp. 207-214.
    CCLRC Daresbury Laboratory.
  2. W. Shepard, M. Ramin, R. Kahn and R. Fourme (1997).
    MASC: A Combination of Multiple-Wavelength Anomalous Diffraction & Contrast Variation.
    Proceedings of the CCP4 Study Weekend, pp. 103-117
    CCLRC Daresbury Laboratory.
  3. A. G. Urzhumtsev, E. A. Vernoslova and A. D. Podjarny (1996).
    Approaches to Very Low Resolution Phasing of the Ribosome 50S particle from Thermus thermophilus by the Few-Atoms-Models and Molecular-Replacement Methods.
    Acta Cryst., D52, 1092-1097
    Synopsis:
    An ab initio crystallographic image of the T50S ribosomal particle is obtained at 80 Å resolution using the few-atoms-models method.
  4. K. M. Andersson and S. Hovmuller (1996).
    Phasing Proteins at Low Resolution.
    Acta Cryst., D52, 1174-1180
    Synopsis:
    A method for obtaining correct phases of low-order reflections for globular proteins using a spherical scattering function is presented. The method predicts the phases of the ten or so lowest order reflections which might enable the determination of a low-resolution envelope of an unknown protein.
  5. C.W. CARTER, K.V. CRUMLEY, D.E. COLEMAN, F. HAGE & G. BRICOGNE (1990).
    Direct phase determination for the molecular envelope of tryptophanyl tRNA synthetase from Bacillus Stearothermophilus by X-ray contrast variation.
    Acta Cryst. A46, 57-68.
  6. A. Urzhumtsev and A. Podjarny (1995).
    On the solution of the molecular-replacement problem at very low resolution: application to large complexes.
    Acta Cryst. D51, 888-895
    Synopsis:
    The applicability of the molecular-replacement method to structure solution at very low resolution is shown, both with atomic and non-atomic (envelope) models. The specific nature of the signal at this resolution required the development of a new protocol for molecular replacement.
  7. V. Yu Lunin, N. L. Lunina, T. E. Petrova, E. A. Vernoslova, A. G. Urzhumtsev and A. D. Podjarny (1995).
    On the ab initio solution of the phase problem for macromolecules at very low resolution: the few atoms model method
    Acta Cryst. D51, 896-903.
    Synopsis:
    The phase problem is solved ab initio at very low resolution for macromolecular through the random generation of a very large amount of few atoms models, the selection of the best ones with an amplitude correlation checking, and the grouping the best models in `clusters'. Applications are described.
  8. Lunin, V.Yu., Urzhumtsev, A.G., Skovoroda, T.A. (1990).
    Direct low-resolution phasing from electron-density histograms in protein crystallography.
    Acta Cryst., A46, 540-544
    The first time cluster analysis was used for the phase problem solution.
  9. Urzhumtsev, A.G. (1991)
    Low-resolution phases: their influence on SIR-syntheses and retrieval with double-step-filtration
    Acta Cryst., A47, 794-801
    The impoirtance of very low resolution data was demonstrated with calculated and experimental data
  10. Podjarny, A., Schevitz, R.W., Sigler, P.B. (1981)
    Phasing low-resolution macromolecular structure factors by matrical direct methods
    Acta Cryst., A37, 662-668
    The first "phase extention" toward LOW resolution
  11. Fourme, R., Shepard, W., Kahn, R., L'Hermite, G. & De La Sierra, I. (1995)
    The Multiwavelength Anomalous Solvent Contrast (MASC) Method in Macromolecular Crystallography
    Journal of Synchrotron Radiation, 2, 36-48.
Contact

For more information concerning low resolution data collection on stations 7.2 and 9.5 contact : James Nicholson or Pierre Rizkallah

James Nicholson
TEL: 01925 603626
E-mail: J.Nicholson@dl.ac.uk
Pierre Rizkallah
TEL: 01925 603388
E-mail: P.J. Rizkallah@dl.ac.uk


Rationale
Technical Issues for 7.2 | Protocol for 7.2 | Experiments on 7.2
Technical Issues for 9.5 | Protocol for 9.5 | Experiments on 9.5
References | Contact

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