Document identifier:
CRISP_D10.1
.doc
Date: 1 November 2012
Authors
E. Mossou, M. Liang, F. Romoli,
M.G. Cuypers, S. McSweeney,
H. Chapman, T. Forsyth
Abstract: Feasibility studies have been carried out to assess the application of common approaches for cryo-freezing, sample humidification for crystals and fibres.
Joint solution scattering methods are being planned in which structural studies exploiting both neutron experiments and analysis will be carried out. Feasibility studies have also been carried out in relation to the use of the Hamburg XFEL source – this was usefully informed by a recent visit of three of the CRISP consortium members to the Stanford SLAC Linear Coherent Light Source (LCLS) facility and has formed the basis of future work that will combine the capabilities and expertise of ILL, ESRF, and XFEL teams.
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1. Cryofreezing feasibility studies
Using a number of model crystal systems, we have devised a system whereby we have investigated the use of a cryostream system commonly used at the ESRF on
ILL’s D19 neutron diffractometer. Critical issues were (i) the ability to locate the cryostream head sufficiently close to the crystal without either obscuring the neutron beam or ending up with the sample too distant from the appropriate part of the stream (ii) the ability to adequately/reliably cool the larger crystals over the extent of the sample (iii) the minimization of ice formation during extended periods of data collection
– see Figure 1.
Offline tests prior to the first D19 cryogenic experiment on crystals were carried out on a number of crystals in order to evaluate the icing problem and attempt to resolve it. A number of key modifications were made and the technique was then adapted online. It was determined that direct freezing of the crystal with the cryostream (instead of prior flash-freezing of the sample in liquid nitrogen) was more efficient for the system studied (Rubredoxin), a different mounting system (a quartz capillary maintaining the crystal by surface tension) was developed
– see Figure 2.
Icing-up of the system was still observed but was successfully dealt with.
Figure 1: Offline D19 tests carried out on a lysozyme crystal. Icing was observed after 4 hours. This picture was taken after 48 hours
Figure 1: New mounting system developed during the experiment
1-20-18 on a rubredoxin crystal
We believe that this part of the work has successfully demonstrated the feasibility of a common approach for cryofreezing of samples that is compatible with joint neutron/X-ray crystallographic measurements.
2. Humidity feasibility studies for crystal and fibre diffraction
The humidity system (HC1) has been devised by the EMBL (Sanchez-Weatherby et
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al . 2009 1 ) and is now under development at the ESRF. The aim of this part of the
CRISP project was to test the feasibility of the HC1 streaming system for complementing measurements on the ESRF and ILL beamlines.
This relative humidity system allows precise studies as a function of humidity to be carried out (see Figure 2). The nozzle contains an inner tube conducting humid air to the sample, which is protected by a surrounding dry airflow (coming from the outer nozzle tube). This device uses a simple system based on the control of the dew point. The air is cooled down to a temperature corresponding to the relative humidity required at the sample position.
The HC1 system is now commonly used at the ESRF as a method to control dehydration of protein crystals in an attempt to enhance diffraction quality. It is one of the best methods developed to control dehydration and has succeeded to improve diffraction quality in several cases.
The HC1 also offers major opportunities for the study of fibrous systems. Many systems that form fibres are highly water sensitive (DNA, polysaccharides, amyloid fibres…) – Russi et al ., 2011 2 . We have carried out experiments on amyloid fibres using the HC1 humidity system and have observed a structural transition at ultra high humidity that has never been observed before (one of the reason probably being that such high levels of humidity are notoriously hard to maintain).
Figure 2: Picture of the HC1 humidity controller set-up at the ESRF with on the left the nosepiece of the system and the sample pin and on the right the controller system in place (Sanchez-Weatherby et al. 2008)
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Figure 3: shows 2 diffraction patterns of an amyloid fibre exposed at different levels of relative humidity (RH). There is a fully reversible structural transition appearing at high humidity (appearance of a low angle reflection at 57 Å) corresponding to an increase in diameter of the amyloid filaments from 27-57 Å
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Another HC1 system has been purchased jointly by ILL/ESRF and this system is dedicated to joint X-ray neutron work of this type. Adaptations have been made to
ILL’s D19 neutron diffractometer that have allowed analogous humidity related experiments to be carried out. A few modifications are however still required in order to make this system fully compatible with neutron use. The typically longer exposure times that are required for neutron experiments as well as the orientation of the humidity stream can cause condensation problems.
Subject to the issues raised above we feel that the use of this system will be possible for common X-ray/neutron approaches using an advanced purposedesigned humidity control system.
3. Combined Neutron/X-ray/XFEL studies
Figure 4: Coherent X-ray Imaging (CXI) instrument at the Stanford SLAC LCLS.
The ILL/ESRF/CFEL teams have focused on the feasibility of using complementary neutron/synchrotron X-ray/FEL measurements on a number of biological problems.
One of these is amyloid
– a topical biological problem relevant to human health. The
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feasibility of such a common approach has been evaluated during meetings of the
CRISP, WP team at ILL, ESRF, DESY, and more recently during an experimental run at the Coherent X-ray Imaging (CXI) instrument at SLAC, LCLS (Figure 5).
Theoretical models have indicated that fibre diffraction from a hard X-ray Free
Electron Laser can resolve the cross-beta structure of amyloid using serial crystallography techniques. During this visit, conditions/feasibility of these types of experiments were established.
1 J. Sanchez-Weatherby, M. W. Bowler, J. Huet, A. Gobbo, F. Felisaz, B. Lavault, R. Moya,
J. Kadlec, R. B. G. Ravelli, and F. Cipriani. Improving di_raction by humidity control: a novel device compatible with x-ray beamlines. Acta Crystallogr D Biol Crystallogr,
65:1237{1246, 2009.
2 S. Russi, D H. Juers, J. Sanchez-Weatherby, E. Pellegrini, E. Mossou ,V. T. Forsyth, J. Huet, A.
Gobbo, F. Felisaz, R Moya, S. M.. McSweeney, S. Cusack, F. Cipriani, M. W. Bowler. Inducing phase changes in crystals of macromolecules: Status and perspectives for controlled crystal dehydration. J.
Struct. Biol. 175(2), 236-243. 2011.
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