Very Low Resolution Data
Stations 7.2 and
Liz Duke &
Daresbury Laboratory - PX Facility Group
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
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
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.
- A synchrotron source so the flux is maintained even for a
finely collimated beam.
- 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.
- The Hamburg PX camera, which features a long
collimator and beamstop translation.
Protocol for Collecting Very Low Resolution
Data on Station 7.2.
- Collimate the beam to 150-100 microns using the slits on the
- Hamburg PX Camera
- Collimate the beam at the monochromator using the pre-mono
- Move the beamstop away from the crystal by ~10cm.
- 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.
|Rotation Range (total)||120o
|Rotation Step ||3o
|Resolution Limits(Å)||200 - 8.5
||185 - 20|
|Unit Cell (Å)||a=130.1,|
|Merging R-Factor (%)||3.5
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.
|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
- A synchrotron source so the flux is maintained even for a finely collimated beam.
- 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).
- The detector can be translated back along the optical bench up to 1400 mm.
- The beamstop can be positioned anywhere between the sample and the detector.
- Pre-collimator slits are available to reduce the beam size.
Collecting Very Low Resolution Data on Station 9.5
- Set the monochromator to the rquired wavelength (0.7 to 1.5 Å).
- Collimate the beam to 100-200 microns using the pre-collimator slits.
- Move the beamstop away from the sample.
- Move the MAR detector back along the optical bench (maximum distance approx. 1400 mm).
- 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
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 pattern collected on station 9.5. The circles correspond to
resolutions of 80, 40, 27 and 20 Å respectively.|
- Urzhumtsev, A., Lunin, V. & Podjarny, A. (1997).
Recent Advances in Phasing,
Proceedings of the CCP4 Study Weekend, pp. 207-214.
CCLRC Daresbury Laboratory.
- 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.
- 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
Acta Cryst., D52, 1092-1097
An ab initio crystallographic image of the T50S ribosomal particle is
obtained at 80 Å resolution using the few-atoms-models method.
- K. M. Andersson and S. Hovmuller (1996).
Phasing Proteins at Low Resolution.
Acta Cryst., D52, 1174-1180
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.
- 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
Acta Cryst. A46, 57-68.
- 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
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.
- 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.
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.
- Lunin, V.Yu., Urzhumtsev, A.G., Skovoroda, T.A. (1990).
Direct low-resolution phasing from electron-density histograms in protein
Acta Cryst., A46, 540-544
The first time cluster analysis was used for the phase problem solution.
- Urzhumtsev, A.G. (1991)
Low-resolution phases: their influence on SIR-syntheses and retrieval
Acta Cryst., A47, 794-801
The impoirtance of very low resolution data was demonstrated with
calculated and experimental data
- Podjarny, A., Schevitz, R.W., Sigler, P.B. (1981)
Phasing low-resolution macromolecular structure factors by matrical
Acta Cryst., A37, 662-668
The first "phase extention" toward LOW resolution
- Fourme, R., Shepard, W., Kahn, R., L'Hermite, G. & De La Sierra, I. (1995)
The Multiwavelength Anomalous Solvent Contrast (MASC) Method in Macromolecular
Journal of Synchrotron Radiation, 2, 36-48.
For more information concerning low resolution data collection
on stations 7.2 and 9.5 contact:-
TEL: 01925 603626
or Pierre Rizkallah.
TEL: 01925 603388
E-mail: P.J. Rizkallah@dl.ac.uk