short communications The Macromolecular Neutron Diffractometer MaNDi at the Spallation Neutron Source ISSN 1600-5767
Leighton Coates,a* Matthew J. Cuneo,a Matthew J. Frost,a Junhong He,a Kevin L. Weiss,a Stephen J. Tomanicek,a Hana McFeeters,b Venu Gopal Vandavasi,a Paul Langana and Erik B. Iversona Received 22 August 2014 Accepted 9 June 2015
a Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37830, USA, and bDepartment of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL 35899, USA. *Correspondence e-mail:
[email protected]
Edited by V. T. Forsyth, Institut Laue–Langevin, France, and Keele University, UK
The Macromolecular Neutron Diffractometer (MaNDi) is located on beamline 11B of the Spallation Neutron Source at Oak Ridge National Laboratory. The instrument is a neutron time-of-flight wavelength-resolved Laue diffractometer optimized to collect diffraction data from single crystals. The instrument has been designed to provide flexibility in several instrumental parameters, such as beam divergence and wavelength bandwidth, to allow data collection from a range of macromolecular systems.
Keywords: MaNDi; Spallation Neutron Source; neutron diffractometers.
1. Introduction Macromolecular neutron crystallography is a powerful tool for determining the positions of hydrogen atoms and water molecule orientations, and for identifying different chemical species. The flux at neutron protein crystallography beamlines is orders of magnitude lower than that at their X-ray counterparts. As a consequence, most of the protein crystals that have been used to collect full neutron data sets have volumes >1 mm3. Recently, a number of instrument upgrades, such as the repositioning of the LADI-III diffractometer at the Institut Laue–Langevin (Blakeley et al., 2010), coupled with developments in protein perdeuteration Hazemann et al., 2005; Meilleur et al., 2009) have enabled much smaller crystals (0.1 mm3) to be used for neutron data collection (Blakeley et al., 2008; Howard et al., 2011; Weber et al., 2013). Several newly constructed neutron beamlines have also recently come online, such as the BIODIFF diffractometer at the FRM2 reactor and the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory (ORNL) (Coates et al., 2010; Meilleur et al., 2013). This has significantly increased the availability of neutron diffraction beamtime to the global user community. In addition, many of these neutron beamlines are now capable of collecting neutron diffraction data at cryogenic temperatures of around 100 K, enabling the study of complex and freeze-trapped systems (Casadei et al., 2014; Coates et al., 2014).
2. The MaNDi instrument
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The neutron optics system design on MaNDi has been detailed previously (Coates et al., 2010). Briefly, MaNDi uses a 24 mlong curved and tapered neutron guide to deliver neutrons to the sample position, which is located 30 m from the neutron moderator. Three bandwidth choppers are located at the start of the instrument to select the wavelengths of neutrons that will be used in the experiment. The neutron guide has a radius
http://dx.doi.org/10.1107/S1600576715011243
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short communications of curvature of 1200 m, providing intrinsic sample protection in the case of an upstream bandwidth chopper failure that might allow harmful fast neutrons or gamma rays to reach the sample position. The last 2.4 m of the neutron guide are interchangeable and are mounted on a neutron optics table. This enables different neutron guide optics to be used in unison with two sets of absorbing slits at each end of the optics table to alter the incident beam divergence at the sample position. An overview of the major beamline components used in the MaNDi beamline is given in Fig. 1. The horizontal and vertical beam divergence at the sample position can be
altered over a large range from 0.80 to 0.12 FWHM (Coates et al., 2010), enabling MaNDi to be used for studying several different kinds of samples. The sample position itself is surrounded by a spherical detector array frame that is currently populated with 30 out of a total of 46 possible Spallation Neutron Source (SNS) Anger camera detectors, with a sample-to-detector distance that varies between 39 and 45 cm, currently giving a detector coverage of 3.3 sr. The sample position itself is not easy to reach because of the large number of detectors that surround the sample. Therefore, the instrument goniometer (Fig. 2) is raised from the top of the detector array frame for sample loading. After the sample has been mounted, the goniometer is translated downwards to interlock with a set of kinematic mounts on top of the data array frame by a motorized goniometer lifting and lowering mechanism. The Crystal Logic (Los Angeles, California) custom goniometer has a built-in Oxford Cryosystems Cobra cryostream for data collection at temperatures between 80 and 200 K.
3. Beamline commissioning data
Figure 1 An overview of the MaNDi instrument. The neutron guide starts at 6 m from the neutron moderator and three bandwidth choppers are present at 6.2, 7.2 and 10.5 m from the moderator. At 24 m from the moderator, a secondary shutter is present to enable safe entry into the instrument. The last 2.4 m of the neutron guide accommodates interchangeable components which are mounted on a neutron optics table. The detector array frame has a total of 46 detector ports, 30 of which are occupied by detectors at the present time. The sample positioner (goniometer) is raised into the sample loading position using the sample positioner lift.
As part of routine beamline commissioning, we performed a series of measurements intended to document the incident neutron beam available to users of the MaNDi instrument and assess the performance of the neutron guide system as it was installed. We used a calibrated neutron beam monitor (Iverson et al., 2006) downstream of the final guide section at 29.57 m flight path length (per survey). The SNS accelerator system was operated for a short period at 5 Hz in order to preclude frame overlap and permit us to test the beam without bandwidth choppers; at 5 Hz and 30 m, we had available to us ˚ . With the MaNDi a neutron bandwidth of approximately 26 A neutron guide arranged in its high-intensity configuration (beam divergence 0.80 FWHM), Monte Carlo simulations using McStas (Willendrup et al., 2004) predict a current exiting the neutron guide as shown in Fig. 3. Also shown in Fig. 3 is the measured neutron current exiting the guide system in the same
Figure 2 (a) The MaNDi sample positioning system is shown in the sample loading position. It is a custom goniometer with 360 rotation on ! and ’ and a fixed of 135 . A motorized goniometer head with x, y, z movement is used to finely position the sample into the neutron beam. The crystal position is visualized using a pair of sample positioning cameras and lights mounted into two spare detector ports. (b) The inside of the detector array frame is shown. The black color inside is caused by a layer of neutron-absorbing 10B powder, which is used to reduce the neutron background. J. Appl. Cryst. (2015). 48, 1302–1306
Figure 3 A plot showing in blue the simulated current exiting the MaNDi neutron guide in the high-intensity configuration. Shown in red is the measured neutron current exiting the guide system in the same configuration. At ˚ ), the predicted and measured currents the spectral peak (around 2.5 A are within 10% of each other. Leighton Coates et al.
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short communications high-intensity configuration. At the spectral peak (around ˚ ), the predicted and measured currents are within 10% of 2.5 A each other. At the longest wavelengths, however, the measured current exceeds predictions by nearly a factor of two. The performance at longer wavelengths is significantly better than predicted; we believe this to be due to guide optics exceeding the original design specifications. It should be noted that the data shown in Fig. 3 are not the ‘flux’; the use of neutron guides implies that the beam spot size will change with changing wavelength, and this set of measurements and calculations considers the entire beam regardless of its extent. Further measurements at 5 Hz, in which each bandwidth chopper was run in isolation, permitted us to calibrate phase settings for those choppers to provide specific neutron wavelength bands. Subsequent measurements at full SNS repetition rate (60 Hz) further provide an estimate of the impact of high proton beam power on the neutron beam performance. At 1 MW operation, the long wavelength intensity per unit power is approximately 20% lower than at reduced (80 kW) powers. We also undertook measurements of the wavelengthdependent incident beam divergence. A detector apparatus developed at SNS (Berry et al., 2012) provides measurement of the full phase space density of the neutron beam. Fig. 4 shows ˚ in the high-intensity the actual beam divergence at 2.5 A configuration.
4. MaNDi 60 and 30 Hz operation As the SNS operates at 60 Hz and the MaNDi sample position is located 30 m from the neutron moderator, the maximum ˚ (Coates et al., 2010) usable neutron bandwidth is = 2.16 A when MaNDi accepts every emitted neutron pulse. The
chopper system on MaNDi can also be run at 30 Hz, in which case every other neutron pulse is rejected. This serves to double the maximum usable neutron bandwidth to = ˚ . This larger neutron bandwidth coupled with high 4.33 A detector coverage enables MaNDi to collect large quantities of diffraction data in each crystal orientation. Using the CrystalPlan software (Zikovsky et al., 2011) and the MaNDi detector array geometry, it is possible to calculate the area of reciprocal space covered in each orientation for a given sample. For a crystal in space group P1, collecting data to a ˚ , the 30 detectors now on MaNDi can cover resolution of 2.0 A 20% of reciprocal space in each orientation using neutrons ˚ for data collection. This with wavelengths between 2 and 6 A means that for favorable hexagonal space groups a complete data set can be collected in as few as three different crystal orientations. This 30 Hz setting is particularly useful for small, weakly diffracting crystals as it allows data collection with a minimal number of orientations while also allowing longerwavelength neutrons to be used for data collection. As the effective flux (Jauch, 1997) increases as the square of the wavelength, data from weakly diffracting crystals (Dmin = 2.5– ˚ ) are better collected using higher neutron wavelengths 3.0 A ˚ ). When using these higher wavelengths for data (2–6 A collection, the higher-resolution reflections will have 2 values well above 90 . However, several complications can arise from the utilization of longer-wavelength neutrons for data collection, such as increased absorption by the sample, that must be carefully weighed up before data collection. The spherical detector array frame on MaNDi (Fig. 5) ensures that these reflections can be measured up to 2 values of 150 when fully populated.
5. Data collection, reduction and refinement MaNDi is able to operate with a neutron bandwidth of ˚ , which can be taken anywhere between 1 and 2.16 or 4.33 A
Figure 5 Figure 4 ˚ as The wavelength-dependent incident beam divergence at 2.5 A measured on MaNDi in the high-intensity 0.80 FWHM setting. A special detector developed at SNS provides measurement of the full phase space density of the neutron beam. The measured FWHM of the neutron beam was 0.86 0.69 .
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The MaNDi detector array frame is shown populated with 30 SNS Anger camera modules. The total number of detector ports is 48. However, two ports are occupied by video cameras with co-axial lights used for sample alignment. This leaves a total of 46 detector ports available for population with SNS Anger camera modules. The data array frame will be fully populated with 46 detectors by the end of 2015. J. Appl. Cryst. (2015). 48, 1302–1306
short communications Table 1 Data statistics from selected samples collected during the commissioning phase of the MaNDi instrument. Values in parentheses correspond to the highest resolution shell.
Degree of deuteration Temperature (K) ˚) Wavelength band (A ˚) Resolution (A No. reflections measured No. unique reflections Completeness (%) No. images Oscillation angle ( ) Exposure time per image (h) Space group ˚) Unit-cell parameters (A Rmerge Rp.i.m. Multiplicity Mean I/(I)
Toho-1 -lactamase
Vitamin B12
tRNA hydrolase
Fully perdeuterated 293 2–6.3 20.61–2.05 (2.12–2.05) 74 602 (4085) 16 800 (1343) 85.5 (69.3) 7 10 6 P3221 a = 74.1, b = 74.1, c = 99.90 0.235 (0.223) 0.106 (0.120) 4.4 (3.0) 8.4 (3.4)
Exchanged in D2O 100 1–3.16 10.08–1.2 (1.24–1.20) 12 467 (810) 2462 (220) 78.1 (71.7) 15 10 4 P212121 a = 15.93, b = 21.79, c = 26.68 0.377 (0.28) 0.149 (0.130) 5.1 (3.7) 7.7 (6.1)
Fully peudeuterated 293 2–4 20.61–2.50 (2.59–2.50) 12 458 (909) 5378 (469) 75.3 (69.2) 5 10 24 P6122 a = 64.93, b = 64.93, c = 156.51 0.299 (0.401) 0.196 (0.293) 2.3 (1.9) 3.8 (3.0)
˚ . For small molecules such as natrolite and vitamin B12, 10 A which diffract to high resolution, MaNDi typically operates in ˚ 60 Hz mode using neutron wavelengths between 1 and 3.16 A to give the highest resolution diffraction data possible. For well diffracting protein crystals such as Toho-1 -lactamase (Coates et al., 2014), MaNDi can operate at 30 or 60 Hz. For smaller, more weakly diffracting macromolecular crystals, the instrument normally operates at 30 Hz using neutron wave˚ . During each exposure, the crystal lengths between 2 and 6 A is kept static and is typically rotated by 5–10 on the ’ axis between exposures. To enable higher levels of data completeness, a second data set can be collected at a different ! angle if required and the two data sets merged. Currently, MaNDi diffraction files are saved in a NeXus format and reduced using the single-crystal routines (Schultz et al., 2014) present in the Mantid software project. Data are then wavelength normalized and scaled using ANDREV (Schultz et al., 1984) or the Lauenorm program from the Lauegen suite (Campbell et al., 1998). Wavelength normalization for Laue data involves the use of symmetry-equivalent reflections measured at different wavelengths to calculate a wavelength normalization curve. This places all data on the same scale regardless of the wavelength they were collected at. The wavelength normalization curve is typically calculated by splitting the data into several wavelength bins, scaling these bins together and curve fitting the resulting scale factors (Campbell et al., 1998). Data merging statistics are generated using the PHENIX suite (Adams et al., 2010) and refinement is normally done using the phenix.refine program or using SHELXL (Gruene et al., 2014).
MaNDI at the time. A time-of-flight slice from part of a diffraction pattern collected from the Toho-1 -lactamase crystal is shown in Fig. 6. The vitamin B12 and Toho-1 -lactamase crystals were large (1.5 and 4 mm3, respectively); however, the tRNA hydrolase crystal was much smaller (0.15 mm3). We originally studied this protein with X-rays (Hughes et al., 2012), and the crystals have a unit-cell axis in ˚ and diffracted X-rays to a resolution of excess of 150 A ˚ 2.0 A at the Advanced Photon Source (Hughes et al., 2012). The data collection statistics for these three commissioning data sets are given in Table 1.
7. Future expansions During the commissioning of the MaNDi instrument, the neutron beam power at the SNS increased from 850 kW to 1.3 MW, close to its planned final power of 1.4 MW. Possible
6. First data sets During the commissioning process, a series of data sets were collected on the MaNDi instrument for testing purposes. These included the structures of vitamin B12 and the perdeuterated protein structures of Toho-1 -lactamase and a tRNA hydrolase, amongst others. All of these structures were collected using the 20 Anger camera detectors installed on J. Appl. Cryst. (2015). 48, 1302–1306
Figure 6 A selected time-of-flight slice corresponding to neutrons with wave˚ from a Toho-1 -lactamase diffraction lengths between 2.8 and 3.0 A pattern. For clarity, the three-dimensional detector orientations of the Anger cameras have been mapped onto a two-dimensional plane and 11 detectors are shown. Leighton Coates et al.
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short communications future power upgrades to the SNS accelerator could raise the beam power to a maximum of 2 MW, which the MaNDi beamline shielding and components were engineered to operate at. Recently an additional ten SNS Anger camera detectors were fitted into the MaNDi detector array frame, bringing the total number of fitted detectors to 30. Sixteen more detectors will be installed by the end of 2015 to fully populate the detector array frame with 46 detectors. This will be more than double the number of detectors that were present during the commissioning of MaNDi.
Acknowledgements This research at ORNL’s Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. The Office of Biological and Environmental Research supported research at Oak Ridge National Laboratory’s Center for Structural Molecular Biology, using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy.
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