Facility and method for molecular structure determination

A molecular structure determination facility includes a first X-ray source capable of emitting a pulsed coherent X-ray beam along a first emission direction and a plurality of first measurement stations aligned along the first emission direction. Each of the first measurement stations comprises a sample injector device for injecting a sample beam of a liquid into an interaction region, a focusing unit for focusing an X-ray beam, and a detector arranged around and comprising a central opening aligned with the emission direction, and being sensitive to X-rays emerging from the interaction region. A method uses the facility by emitting a coherent X-ray beam pulse using the first X-ray source, triggering the injector devices to inject sample beams of liquid into the interaction regions such that the coherent X-ray pulse intersects the sample beams of liquid in the interaction regions, and detecting X-rays emerging from the interaction regions using the detectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority of European Patent Application Number EP 11185746.2, which was filed on Oct. 19, 2011. The disclosure of the above application is incorporated herein by reference in its entirety.

FIELD

The invention relates to a facility and a method for molecular structure determination using coherent X-rays.

BACKGROUND

The structure of biological macromolecules is primarily detected using X-ray crystallography where the diffraction patterns of coherent X-rays interacting with crystallized biological macromolecules is recorded. To obtain highly resolved representations of the molecule's structure these diffraction patterns have to be recorded at wide angles where diffraction intensities are very low. The diffraction intensity is proportional to the number of diffracted X-rays and the number of unit cells in the illuminated single crystal. Therefore, in order to get sufficient diffraction intensities large crystals have to be irradiated with large numbers of photons.

The number of diffracted photons can easily be increased by extending the irradiation time. Unfortunately, many crystals only tolerate dose up to 30 MGy (3×107 J/kg) before substantial structural damages occur that destroy the crystalline structure. The dose required to obtain sufficient diffracted intensities can be reduced by growing larger crystals with more unit cells. For large macromolecules such as proteins and protein complexes staying below the tolerable dose requires crystals of many hundreds of micrometers in size. Growing crystals of this size and high quality is a very difficult and time consuming trial-and-error process that is currently the major bottleneck of X-ray crystallography. Even more challenging are membrane-bound proteins that are critical for drug design but notoriously difficult to crystallize.

The crystallization bottleneck can be overcome by using serial femtosecond crystallography. Here, nanocrystals are not irradiated with conventional synchrotron radiation but a collection of nanocrystals are irradiated one at a time with ultra-short coherent X-ray pulses. This method bears several advantages as the nanocrystals can be more easily grown and substantially higher doses can be employed.

Nanocrystals of many macromolecules can be grown by driving a protein suspension into supersaturation. If the proteins in the supersaturated suspension are quickly precipitated many small nanocrystals are formed around many nucleation sites.

The ultra-short coherent X-ray pulses are commonly generated with X-ray free electron lasers (X-ray FELs) and have a pulse length of approximately 100 fs (10-13 s). If the X-ray FEL pulse is focused to micrometer dimensions it can deposit doses in a crystal that exceed those conventionally tolerated by several magnitudes. As expected, the high dose of the X-ray pulse completely vaporizes the nanocrystal but only after the pulse has passed through it. The short pulse “outruns” the radiation damage as the inertia of the atoms in the crystal is sufficiently large to keep their movements within tolerable bounds during the time that the beam passes through the crystal. Hence, the diffraction pattern that is recorded on the detector corresponds to the undamaged crystal structure.

A single pulse does of course only give a diffraction pattern of the crystal structure in one particular orientation. In order to reconstruct the full three-dimensional structure of the molecule diffraction patterns obtained under many orientations have to be combined. Unlike conventional powder diffraction crystallography the data from many crystals is not summed up without regard to their orientation. Instead each diffraction pattern is indexed i.e. the observed peak intensities are labeled according to their origin in the lattice of the crystal. Those peaks that carry the same index are then summed up. The summation therefore averages over crystal shapes, crystal sizes and crystal orientation. Due to their small size the crystals are coherently illuminated which, combined with the index summation, leads to brighter intensities than those obtained with conventional crystallography on large crystals. It is therefore expected that more information can be extracted. It may, for example, be possible to extract a three dimensional vector gradient of the intensities which would increase the measured information by a factor of four. This would allow the use of novel phasing methods to obtain the molecular structure of the macromolecule.

In total, diffraction patterns from more than 10,000 crystals have to be measured and summed up. In these experiments approximately 10% of the X-ray pulses hit a nanocrystal. Out of the recorded diffraction patterns roughly half can be indexed successfully. Therefore, a total of 200,000 X-ray pulses is required to obtain sufficient data for a complete reconstruction of the structure of the macromolecule. Current X-ray FELs achieve a repetition rate of 120 Hz i.e. for each macromolecule at least 28 minutes of beam time is required.

SUMMARY

In the view of the foregoing discussion it is therefore the object of the present invention to provide a facility and a method for molecular structure determination that reduces the time that is required to determine the structure of a single molecule significantly and thereby drastically increases the throughput of molecules that can be studied using a single X-ray FEL.

According to a first aspect of the present invention a molecular structure determination facility comprises a first X-ray source capable of emitting a pulsed coherent X-ray beam along a first emission direction, a plurality of first measurement stations aligned consecutively along the first emission direction, wherein each of said first measurement stations comprises a sample injector device for injecting a sample beam of liquid into an interaction region located on said first emission direction, a focusing unit for focusing an X-ray beam in a focal spot located in said interaction region and a detector being sensitive to X-rays emerging from said interaction region, said detector being arranged around said first emission direction and on that side of said interaction region facing away from said X-ray source, wherein said detector comprises a central opening aligned with said emission direction.

Thus, the present invention provides a solution for the above problem by providing a molecular structure determination facility comprising a first X-ray source capable of emitting a pulsed coherent X-ray beam along a first emission direction. This X-ray source may be but is not limited to an undulator employed in an X-ray Free Electron Laser (X-ray FEL).

Along the first emission direction a plurality of first measurement stations is consecutively aligned so that the X-ray beam may pass through the stations. Each of said first measurement stations comprises a sample injector device for injecting a sample beam of a liquid into an interaction region located on said first emission direction. The injector device may, for example, provide a gas-focused aerosol jet of a suspension carrying the nanocrystals, a continuous liquid water stream carrying the nanocrystals or a pulsed stream of liquid carrying the nanocrystals. A pulsed stream of liquid may, for example, be injected at the same rate as the X-ray pulses arriving at the measurement station. Thereby, the consumption of the sample suspension is reduced, since less material will be flowing overall, yet the material will be flowing during the time that X-ray pulses intersect the interaction region. This embodiment is especially advantageous when the sample suspension can only be obtained in limited volumes.

Said liquid may, for example, be a suspension of nanocrystals in liquid, a solution of uncrystallized protein macromolecules or other biological objects such as complexes. Uncrystallized protein macromolecules will give rise to less information regarding the scattering pattern but could be useful in time-resolved experiments where the initial structure is well known. It would be possible, for example, to determine the change of structure under illumination with a visible or an IR laser or after mixing of two proteins.

Said first measurement stations further comprise a focusing unit for focusing an X-ray beam in a focal spot located in said interaction region. The X-ray beam could, for example, be focused using a compound refractive lens or grazing-incidence curved-mirror optics such as Kirkpatrick-Baez mirrors.

Additionally, said first measurement stations comprise a detector being sensitive to X-rays emerging from said interaction region. Said detector is arranged around said first emission direction and on that side of said interaction region facing away from said X-ray source. Said detector comprises a central opening aligned with said emission direction. Advantageously, said detector comprises a set of two low-noise, X-ray p-n junction charge-coupled device (pnCCD) modules or high-repetition rate pixel-array detectors.

The present invention provides a possible way of increasing the throughput of molecular structure determination facility without requiring multiple pulsed X-ray sources.

The invention essentially proceeds from the fact that less than 1% of the incident X-ray beam pulse's energy is absorbed when the beam interacts for the first time with a sample. More than 99% of the incident X-ray beam pulse's energy is transmitted undiffracted through the sample and passes through said central opening in said detector. Thereby, the detector is spared from severe damage by the X-ray beam and the X-ray beam can be utilized for further diffraction measurements in said consecutively aligned measurement stations.

Upon entering the measurement station closest to the X-ray beam source the X-ray beam is focused to a focal spot within the interaction region of the first station. In this first focal spot a focal width of 0.1 to 0.5 micrometer could be achieved, for example. Subsequently, the beam is divergent once it has passed the focal spot. Upon entering the next consecutively aligned measurement station the beam is refocused to a focal spot within the interaction region of said next consecutively aligned measurement station. By way of example a focal width of 1 micrometer could be achieved. Consequently, the beam has to be refocused again in subsequent measurement stations where, for example, a focal width of 2 to 3 micrometers could be expected. The intensity of the X-ray beam decreases with increasing spot size and therefore measurement stations nearer to the X-ray source have higher X-ray beam intensities.

By way of example, all measurement stations can be used to analyze the same macromolecule which reduces the time required to obtain sufficient data for a full 3D reconstruction by a factor equal to the number of measurement stations. In this case any of the injector devices are supplied with the same macromolecule suspension.

By another way of example, nanocrystals from different macromolecules can be screened at each measurement station. The measurement station closest to the X-ray beam source provides the smallest focal width and hence highest intensity of the X-ray beam. It may, for example, be used to obtain diffraction data for very small nanocrystals or provide diffraction data to reconstruct highly resolved structures. However, the high intensities of the measurement station nearest to the X-ray beam source may not be required for all nanocrystals. These nanocrystals could as well be studied with sufficient resolution at the other measurement stations.

In another exemplary fashion, any sample suspension of nanocrystals could first be analyzed in the measurement station with the lowest X-ray beam intensity. If the intensities of the recorded diffraction patterns turn out to be too weak the sample is analysed at another measurement station with a higher X-ray beam intensity.

Alternatively, one or more of the measurement stations with lower X-ray beam intensities could be used for screening measurements to determine whether nanocrystals diffract at all. Nanocrystal suspensions from a range of preparation conditions can be tested in series to determine which preparation condition gives the highest diffraction intensities. The selected suspension can then be used in a measurement station with a higher intensity for structure determination measurements.

In a preferred embodiment, a first X-ray beam analysing device is located on said first emission direction on that side of said first measurement stations facing away from said first X-ray source. Said first X-ray beam analysing device may be used to optimize X-ray beam characteristics. The focusing of the X-ray beam by said focusing units comprised in said first measurement stations or the position of the first emission direction relative to the sample beams of liquid can be monitored by the first X-ray beam analysing device, for example.

It is particularly advantageous for the facility to comprise a second X-ray source for emitting a pulsed coherent X-ray beam along a second emission direction. Along said second emission direction a plurality of second measurement stations is consecutively aligned. Each of said second measurement stations comprises a sample injector device for injecting a sample beam of a liquid into an interaction region located on said second emission direction, a focusing unit for focusing an X-ray beam in a focal spot located in said interaction region and a detector being sensitive to X-rays emerging from said interaction region. The detector is arranged around said second emission direction on that side of said interaction region facing away from said second X-ray source. Said detector comprises a central opening aligned with said second emission direction.

A second X-ray source emitting along a second emission direction is especially advantageous as it further increases the number of suspension samples that can be studied in the facility. The sampling rate of a single emission direction is limited by the repetition rate of current X-ray FELs that is, for example, at 120 Hz. It is further limited by the read-out frequency of the detectors which is currently, for example, at 200 Hz and the rate with which the injector devices could supply a macromolecule suspension samples to the interaction region. Therefore, a second array of X-ray source and measurement stations would double the throughput of the molecular structure determination facility.

It is again preferred that a second X-ray beam analysing device is located on said second emission direction on that side of said second measurement stations facing away from said second X-ray source. Said second X-ray beam analysing device may be used to optimize X-ray beam characteristics. The focusing of the X-ray beam by said focusing units comprised in said second measurement stations or the position of the second emission direction relative to the sample beams of liquid can again be monitored.

In a preferred embodiment, a source of a pulsed electron beam is provided for supplying a pulsed electron beam to said first and second X-ray sources. Said source of said pulsed electronbeam may, for example, be a linear electron accelerator, an electron synchrotron or a combination of the two. Additionally, a switching magnet is provided having an input connection and first and second output connections. Said switching magnet is capable of diverting a pulsed electron beam entering the magnet via said input connection to either of said first and second output connections. Said input connection is connected with said source of a pulsed electron beam, said first output connection is connected with said first X-ray source and said second output connection is connected with said second X-ray source.

An advantage of the latter embodiment is the use of said single source of said electron beam to provide both X-ray sources with electrons. Electron sources that provide sufficiently high energies for X-ray FEL are very large and costly to build and maintain. Their power consumption is also considerably high. Undulators on the other hand are compared to the accelerator small and cheap. Novel accelerators may, for example, provide electron pulses with a repetition rate of up to 10 kHz. An increase of the read-out frequency of the detectors to 1 kHz from today's 200 Hz would allow for a single electron source to provide electrons for up to 10 lines of X-ray sources and measurement stations. Thereby, the throughput of the facility is considerably increased without requiring another electron source.

It is particularly advantageous for the facility to comprise a central sample preparation unit to which said injector devices are connected. Said central sample preparation unit comprises a reservoir system including a plurality of reservoirs, a plurality of pumps and a switching unit. Each of said reservoirs is connected with an input of one of said pumps. The switching unit has a plurality of inputs and a plurality of outputs, wherein the output of each of said pumps is connected with an input of said switching unit and said outputs of the switching unit are connected with the injector devices.

A sample preparation unit as described above could, for example, automatically carry out the different operation modes of the facility that have been described above. Additionally, the sample preparation could be connected to a feedback system that provides information about the diffraction patterns recorded in the measurement stations. Thereby, the flow rate of the sample suspensions or even the preparation of the sample suspensions could be controlled. The sample preparation system could also be used to automatically clean the injection devices with a neutral liquid in between two different sample suspensions.

In a preferred embodiment of the present invention one or a plurality of said measurement stations further comprises a second sample injector device for injecting a second sample beam of liquid into said interaction region. Two macromolecule suspensions could be simultaneously injected into the interaction region such that they mix. If these macromolecules are uncrystallized a reaction may be initiated which changes the structure of the molecules and also the diffraction patterns of the macromolecules. Thereby, a molecular structure determination facility of the preferred embodiment can be used to study the changes of molecular structures within a reaction. It is further possible, for example, to obtain diffraction patterns at different points in time after the beginning of the mixing of the sample beam of liquid and the other sample beam of liquid. Thereby, the structural changes of the molecules in the suspension due to the reaction can be monitored in a time-resolved fashion. It is also conceivable that a measurement station according to this preferred embodiment may be used in different assemblies than the present invention. In particular, the aforementioned independently inventive concept of a measurement station having a sample injector device and a second sample injector device could be used in a molecular structure determination facility comprising a sole measurement station.

In another preferred embodiment one or a plurality of said measurement stations further comprise a laser device emitting a laser beam intersecting said interaction region. The laser device could be emitting, for example, a visible or an infrared laser beam. Such a laser beam could be used to initiate a photo reaction in the macromolecules in the sample beam of liquid wherein the structure of the macromolecules in the suspension changes. Thereby, a molecular structure determination facility of the preferred embodiment can be used to study the changes of molecular structures within a photo reaction. It is further possible, for example, to obtain diffraction patterns at different points in time after the initiation of the photo reaction. Thereby, the structural changes of the molecule due to the photo reaction can be monitored in a time-resolved fashion. It is conceivable that a measurement station according to this preferred embodiment may be used in different assemblies than the present invention.

In a preferred embodiment, one or a plurality of said measurement stations further comprise a backscatter detector being sensitive to X-rays emerging from said interaction region, said backscatter detector being arranged around said first emission direction and on that side of said interaction region facing away from said detector, wherein said backscatter detector comprises a central opening aligned with said emission direction. A combination of the diffraction patterns detected by the detector and the backscatter detector is preferable as it allows reconstructing the structure of the molecules with a resolution of up to half the wavelength of the coherent X-ray beam. Advantageously, said backscatter detector comprises a set of two low-noise, X-ray p-n junction charge-coupled device (pnCCD) modules or high-repetition rate pixel-array detectors.

According to a second aspect of the present invention a method is provided that uses the above described facility and comprises the following steps of emitting a coherent X-ray beam pulse along said first emission direction using said first X-ray source, triggering said injector devices provided in said first measurement stations to inject sample beams of liquid into said interaction regions aligned along said first emission direction such that said coherent X-ray pulse intersects said sample beams of liquid in said interaction regions aligned along said first emission direction, and detecting X-rays emerging from said interaction regions using said detectors provided in said first measurement stations.

A preferred embodiment of the method additionally comprises the steps of emitting a coherent X-ray beam pulse along said second emission direction using said second X-ray source, triggering said injector devices comprised in said second measurement stations to inject sample beams of liquid into said interaction regions aligned along said second emission direction such that said coherent X-ray pulse intersects said sample beams of liquid in said interaction regions aligned along said second emission direction, and detecting X-rays emerging from said interaction regions using said detectors comprised in said second measurement stations.

In another preferred embodiment of the method according to the present invention the step of triggering said injector device to inject the sample beam of liquid into said interaction region further comprises triggering said second injector device to inject said second sample beam of liquid into said interaction region such that said sample beam of liquid and said second sample beam of liquid mix in said interaction region and such that said coherent X-ray beam pulse intersects said sample beam of liquid mixed with said second sample beam of liquid in said interaction region.

It is further preferred that said step of triggering said injector devices to inject the sample beam of liquid into said interaction region further comprises triggering said laser device to emit the laser beam, such that said laser beam intersects said sample beam of liquid in said interaction region comprised in said measurement station.

It is further preferred that said step of triggering said injector devices to inject the sample beam of liquid into said interaction region further comprises triggering said laser device to emit the laser beam, such that said laser beam intersects said sample beam of liquid mixed with said second sample beam of liquid in said interaction region comprised in said measurement station.

Said methods are advantageous for the same reasons that have already been stated above for the claimed molecular structure determination facility.

DETAILED DESCRIPTION

FIG. 1shows the general structure of a preferred embodiment of a molecular structure determination facility1according to the present invention. A source3of a pulsed electron beam5is connected with an input connection7of a switching magnet9. Said switching magnet9comprises in this preferred embodiment ten output connections11ato11j.

The molecular structure determination facility1further comprises ten measurement lines13ato13j. Moreover it is conceivable that the molecular determination facility1comprises more or less than ten measurement lines13ato13j.

Each measurement line13ato13jcomprises an X-ray source15ato15jemitting a pulsed coherent X-ray beam17ato17jalong a linear emission direction19ato19j. The input21ato21jof each X-ray source15ato15jis connected with an output connection11ato11jof the switching magnet9.

In this preferred embodiment within each measurement line13ato13jthree measurement stations23ato23j,25ato25jand27ato27jare aligned consecutively along the emission direction19ato19jbut it is also within the scope of the present invention that each measurement line13ato13jcomprises more or less than three measurement stations23ato23j,25ato25jand27ato27j.

An X-ray beam analysing device29ato29jis located on the emission direction19ato19jon that side of the measurement stations23ato23j,25ato25jand27ato27jthat is facing away from the X-ray source15ato15j, i.e. at the end of each measurement line13ato13jremote from the X-ray source15ato15j. In a preferred embodiment the X-ray beam analysing device29ato29jcomprises means for X-ray beam wavefront diagnostics.

The molecular structure determination facility1further comprises a schematically drawn sample preparation unit31. Conduits33,35,37connect the sample preparation unit31with the measurement stations23ato23j,25ato25jand27ato27j. Conduits33,35,37comprise discrete conduits connecting each measurement station23ato23j,25ato25jand27ato27jindependently with the sample preparation unit31such that each measurement station23ato23j,25ato25jand27ato27jcan be supplied independently with macromolecule suspensions. Discrete conduits to each measurement station23ato23j,25ato25jand27ato27jhave been omitted for the sake of clarity inFIG. 1but are indicated inFIG. 2.

A detailed drawing of a measurement line13acomprising three measurement stations23a,25a,27aand the central sample preparation unit31is shown inFIG. 2. It is conceivable thatFIG. 2shows any of the measurement lines13ato13j. The measurement stations23a,25a,27aare consecutively aligned on the emission direction19aof the X-ray source15a. Each measurement station23a,25a,27acomprises a sample injector device39,41,43for injecting a sample beam of liquid45,47,49into an interaction region51,53,55located on the emission direction19a. The sample beam of liquid is collected in sample beam dumps57,59,61. In a preferred embodiment of the present invention the sample beam of liquid45,47,49collected in said sample beam dump57,59,61is recycled. Thereby, any crystals or molecules that have not been previously hit by an X-ray pulse can be re-injected either in the same or another measurement station23ato23j,25ato25jand27ato27j.

Each measurement station23a,25a,27afurther comprises a focusing unit63,65,67for focusing an X-ray beam17ain a focal spot69,71,73located in said interaction region51,53,55. Within each measurement station23a,25a,27aa detector75,77,79that is sensitive to X-rays81,83,85emerging from said interaction region51,53,55is arranged around said emission direction19a. The detector75,77,79is located on that side of the interaction region51,53,55facing away from the X-ray source15a. Furthermore, the detector75,77,79comprises a central opening87,89,91aligned with the emission direction19a.

The measurement line13afurther comprises an X-ray beam analysing device29alocated on the emission direction19aon that side of the measurement stations23a,25a,27afacing away from the X-ray source15a.

Said sample preparation unit31comprises a reservoir system93including three reservoirs95ato95c. Each of the reservoirs95ato95cis connected with an input97ato97cof one of three pumps99ato99c. The outputs101ato101cof the pumps99ato99care connected to the inputs103ato103cof a switching unit105. The outputs107,109,111of the switching unit105are each connected with one of the sample injector devices39,41,43via separate conduits. The switching unit109comprises additional outputs each connected to one of the measurement stations23bto23j,25bto25jand27bto27j. For the sake of clarity these output are not shown inFIG. 2. It is also conceivable that the reservoir system31comprises more or less than three reservoirs95ato95cand pumps99ato99c.

Said measurement line13afurther comprises three feedback systems113,115,117. Each feedback system113,115,117connects one of the detectors75,77,79with the sample preparation system31.

A second embodiment of a measurement station119according to the present invention is shown inFIG. 3. Even though this embodiment is described as being part of an assembly of a plurality of measurement stations23bto23j,25bto25jand27bto27jbeing aligned on an emission direction19ato19j, it is also possible that this independently inventive concept of a measurement station119is employed as a sole measurement station119in a molecular structure determination facility1. Said measurement station119comprises a sample injector device121for injecting a sample beam of liquid123into an interaction region125located on the emission direction127of an X-ray source (not shown). It further comprises a second sample injector device129for injecting a second sample beam of liquid131into the interaction region125. The sample beam injector121and the second sample beam injector129are connected to a central sample preparation unit31. The sample preparation unit31and connections to the sample preparation unit31have been omitted inFIG. 3for reasons of clarity. It is conceivable that the sample beams of liquid123,131are injected by the sample injector devices121,129simultaneously or one at a time. The sample beams of liquid123,131are collected in a sample beam dump133. In a preferred embodiment of the present invention the sample beams of liquid123,131collected in said sample beam dump133are recycled.

The measurement station119further comprises a laser device135emitting a laser beam137that intersects the interaction region125. In an exemplary fashion said laser device135may be emitting a laser beam137of visible light or infrared light.

Additionally, the measurement station119comprises a focusing unit139for focusing an X-ray beam141in a focal spot143located in said interaction region125. Within the measurement station119a detector145that is sensitive to X-rays147emerging from said interaction region125is arranged around said emission direction127. The detector145is located on that side of the interaction region125facing away from the X-ray source (not shown). Furthermore, the detector145comprises a central opening149aligned with the emission direction127.

Furthermore, the measurement station119comprises a backscatter detector151that is sensitive to X-rays153emerging from said interaction region125. The backscatter detector151is arranged around said emission direction127on that side of the interaction region125facing away from the detector145. Additionally, the backscatter detector151comprises a central opening155aligned with the emission direction127.

It is conceivable that the measurement station119according toFIG. 3may not comprise the laser device135and/or the backscatter detector151and/or may only comprise one of the injector devices121,129. It is further conceivable that one, a plurality or all of the measurement stations23bto23j,25bto25jand27bto27jare formed according to the alternative preferred embodiment shown inFIG. 3.

According to a preferred embodiment of the present invention the molecular structure determination facility1can be used as follows. The source3of a pulsed electron beam5emits an electron pulse157of a pulsed electron beam5. The electron pulse103enters the switching magnet9through the input connection7. The switching magnet9diverts the electron pulse157to one of the ten measurement lines13ato13j. The electron pulse157leaves the magnet through that one of the ten output connections11ato11jthat is connected with the input21ato21jof the X-ray source15ato15jof the one measurement line13ato13j.

When the electron beam119enters the X-ray source15ato15jit emits a coherent X-ray pulse159ato159jof the pulsed coherent X-ray beam17ato17jalong the emission direction19ato19j. The coherent X-ray pulse159ato159jtravels through the measurement stations23ato23j,25ato25j,27ato27jthat are aligned along the emission direction19ato19jbefore it interacts with the X-ray beam analysing device29ato29j. The focusing of the X-ray beam by the focusing units63,65,67comprised in the measurement stations23ato23jor the position of the emission direction19ato19jrelative to the sample beams of liquid45,47,49can be monitored by the X-ray beam analysing devices29ato29j.

Upon entering a measurement station23ato23j,25ato25j,27ato27jthe coherent X-ray pulse159ato159jis focused in the focal spot69,71,73located in said interaction region51,53,55using the focusing unit63,65,67. Within each measurement station23ato23j,25ato25j,27ato27jthe provided injector device39,41,43is triggered to inject a sample beam of liquid45,47,49into the interaction regions51,53,55such that the coherent X-ray pulse159ato159jintersects the sample beam of liquid45,47,49in said interaction region51,53,55. Said sample beam of liquid45,47,49may, for example, be injected at the same rate as the coherent X-ray pulses159ato159jarrive at the measurement station23ato23j,25ato25j,27ato27j. Thereby, the consumption of the sample suspension is reduced, since less material will be flowing overall, yet the material will be flowing during the time that X-ray pulses159ato159jintersect the interaction region51,53,55. This embodiment is especially advantageous when the sample suspension can only be obtained in limited volumes. X-rays81,83,85emerging the interaction region51,53,55after the coherent X-ray pulse159ato159jhas intersected the sample beam of liquid45,47,49are detected using the detector75,77,79.

Once the coherent X-ray pulse159ato159jhas passed the interaction region51,53,55it leaves the measurement station23ato23j,25ato25j,27ato27jthrough the central opening87,89,91in the detector75,77,79.

In a further preferred embodiment the switching magnet9diverts the electron pulse157to another one of the ten measurement lines13ato13j. The electron pulse157leaves the magnet through that one of the ten output connections11ato11jthat is connected with the input21ato21jof the X-ray source15ato15jof the other measurement line13ato13j.

According to another preferred embodiment of the present invention the source3emits electron beam pulses119at a given repetition rate. Said switching magnet diverts each electron pulse157in succession to one of said measurement lines13ato13jsuch that each X-ray sources15ato15jemits X-ray pulses159ato159jat a repetition rate equal to the repetition rate of the source3reduced by a factor equal to the inverse of the number of measurement lines13ato13j. Therefore, the maximum required read-out frequency of the detectors75,77,79and the maximum required repetition rate of the sample injector devices39,41,43only has to be equal to the repetition rate of the source3reduced by a factor equal to the inverse of the number of measurement lines13ato13jand not equal to the repetition rate of the source3. Likewise, the rate at which the sample beam of liquid45,47,49interacts with the coherent X-ray beam17ato17jand those X-rays81,83,85created in said interaction are detected by the detectors75,77,79can be increased by a factor equal to the number of measurement lines compared to another preferred embodiment in which the electron pulse5is only diverted to one measurement line13ato13j.

A molecular structure determination facility1according to the above exemplary embodiment has a significantly higher throughput than conventional X-ray crystallography experiments. In an exemplary embodiment a molecular structure determination facility1comprises ten measurement lines13ato13jeach comprising two measurement stations23ato23j,25ato25j,27ato27j. The source3emits electron pulses119at a frequency of 10 kHz that are evenly distributed among the measurement lines13ato13jusing the switching magnet9. Hence, every X-ray source15ato15jemits coherent X-ray pulses159ato159jat a repetition frequency of 1 kHz. This corresponds to the readout frequency of the detectors.

For each macromolecule diffraction patterns from more than 10,000 crystals have to be measured and summed up. Approximately 10% of the X-ray pulses159ato159jhit a nanocrystal. Out of the recorded diffraction patterns roughly half can be indexed successfully. Therefore at total of 200,000 X-ray pulses159ato159jis required to obtain sufficient data to fully reconstruct a macromolecule. A particular measurement station23ato23j,25ato25j,27ato27jcan therefore measure sufficient diffraction patterns in200s.

If between two types of suspension that are used as sample beams of liquid45,47,49the sample injection devices39,41,43are flushed with a cleaning suspension for 3 minutes the molecular structure determination facility1would achieve an output of 200 samples per hour. If the molecular structure determination facility1runs with a downtime of 50% a total of 2,400 molecules can be analysed per day. Within one month approximately 70,000 different samples could be studied which compares to the total number of structures that have been publicly released within the last forty years.

In another exemplary embodiment of the present invention a molecular structure determination facility1comprising a measurement station119with two sample injector devices121,129as described before with reference toFIG. 3can be used as follows, where only those steps are described that differ from those stated above.

Within a measurement station119the injector device121and the second injector device129are triggered to simultaneously inject a sample beam of liquid123and a second sample beam of liquid131into the interaction region125where the sample beams of liquid123,131mix and a reaction is initiated. The sample beams of liquid123,131are injected such that the coherent X-ray pulse161intersects the sample beam of liquid123mixed with the second sample beam of liquid131in said interaction region125. X-rays127emerging the interaction region125after the X-ray pulse161has intersected the sample beams of liquid123,131are detected using the detector145. In such a way the molecular structure determination facility1according to the present invention may be used to monitor the change of the shape of a biological macromolecule undergoing a reaction with another biological macromolecule. If diffraction patterns are obtained at different points in time after the mixing of the sample beams of liquid123,131a molecular structure determination facility1of the preferred embodiment could be used to study the structural changes of the macromolecules in a time-resolved fashion.

A molecular structure determination facility1comprising a measurement station119with a laser device135could be used in the following exemplary fashion where only those steps are described that differ from the above description. Upon injection of the sample beams of liquid123,131the laser device135may be triggered to emit a laser beam137that intersects the sample beams of liquid123,131in the interaction region125. Said laser device may be, for example, emitting a visible or an infrared light. Thereby, the laser beam137may drive the macromolecules comprised in said sample beams of liquid123,131into a photo reaction. The change of the structure of the macromolecule due to the photo reaction can then be studied in the refraction patterns detected by the detectors145. If diffraction patterns are obtained at different points in time after the initiation of the photo reaction a molecular structure determination facility1of the preferred embodiment could be used to study the structural changes of the macromolecules in a time-resolved fashion.

In another exemplary embodiment of the present invention a molecular structure determination facility1comprising a measurement station119with a backscatter detector151as described before with reference toFIG. 3could be used in the following exemplary fashion where only those steps are described that differ from the above description. After the coherent X-ray pulse161has been focused in the focal spot143located in said interaction region125using the focusing unit139it passes through the central opening155of the backscatter detector151. Additional X-rays153emerging the interaction region125after the X-ray pulse161has intersected the sample beam of liquid131,137are detected using the backscatter detector151. A combination of the diffraction patterns detected by the detector145and the backscatter detector151allows reconstructing the structure of the molecules with a resolution of up to half the wavelength of the X-ray pulse161.