EXPERIENCE WITH THE USE OF A NEUTRON PROTEIN DATA-COLLECTION FACILITY EQUIPPED WITH A LINEAR DETECTOR

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EXPERIENCE WITH THE USE OF A NEUTRON PROTEIN DATA-COLLECTION FACILITY EQUIPPED WITH A LINEAR DETECTOR

A. Wlodawer, L. Sjölin

To cite this version:

A. Wlodawer, L. Sjölin. EXPERIENCE WITH THE USE OF A NEUTRON PROTEIN DATA-

COLLECTION FACILITY EQUIPPED WITH A LINEAR DETECTOR. Journal de Physique Col-

loques, 1986, 47 (C5), pp.C5-115-C5-121. �10.1051/jphyscol:1986515�. �jpa-00225832�

JOURNAL D E PHYSIQUE

Colloque C5, supplement au n o 8, Tome 47, aoiit 1986

EXPERIENCE WITH THE USE OF A NEUTRON PROTEIN DATA-COLLECTION FACILITY EQUIPPED WITH A LINEAR DETECTOR

A. WLODAWER and L. SJ~LIN'

Centre for Chemical Physics, National Bureau of Standards, Gaithersburg MD 20899, U. S. A.

'~epartment of Inorganic Chemistry, Chalmers Polytechnic, Gothenburg, Sweden

RGsumB

-

Un appareillage d'acquisition de donnees neutroniques de proteines equip6 d'un detecteur lin6aire a fonctionnk pendant environ

5

ans et a bt6 utilis6 pour mesurer des donnees de diffraction de cristaux de trois protgines. Des mkthodes informatiques ont kt6 developp6es pour integrer les intensites des reflexions en utilisant un procedk de masque dynamique, pour mesurer et appliquer une correction d'absorption et pour affiner la matrice d'orientation. Les donnees sont de qualit6 acceptable, avec un facteur d'accord

R

symetrique compris entre 0.04 et 0.12. Les donnees ont it6 utili- sees dans plusieurs determinations de structure avec succ&s.

Abstract

-

A neutron protein data collection facility equipped with a linear detector has been in operation for about

5

years and has been used to measure diffraction data from crystals of three proteins. Computational methods have been developed for integrating reflection intensities using a dynamic mask procedure, 'for measuring and applying an absorption correction and for im- proving the orientation matrix. The data are of acceptable quality, with symmetry

R

factors ranging from 0.04 to 0.12. These data were used in several successful structure determinations.

I

-

INTRODUCTION

One- and two-dimensional electronic position-sensitive detectors have become in the last two years the preferred means of data collection in macromolecular crystal- lography laboratories. A number of such devices have been constructed for both

X-

ray and neutron applications, since they are generally capable of increasing the rates of data collection by between one and two orders of magnitude compared to single counter diffractometers. Even more important is the reported improvement in the quality of collected data, as documented, for example, by Howard et al. /I/.

These results are due to several factors: a) Multiple recording of each reflection b) Sophisticated background and peak integration algorithms c) Better signal-to- noise ratio and lower noise than in film methods and d) decreased radiation damage and the ability to collect data from fewer crystals, due to the increased speed of data collection. The latter two factors are, of course, only important for the

X-

radiation, but without the added speed of position-sensitive detectors neutron diffraction studies of macromolecules would not be generally feasible.

It was the need to collect neutron diffraction data from macromolecular crystals as quickly as possible that was the driving force behind the design and construction of the NBS facility. The flux of neutrons on the sample was measured as 6x10' neutrons cm-2 sec-I at TO MW reactor power (recently the flux was doubled due to power in- crease to 20

MW).

This is still

5

to 10 times lower than the flux reported

at

Brookhaven and at the Institute Laue-Langevin /2/ and it was clear that the time re- quired to collect high resolution protein data would be prohibitively long. As will be shown below, modifying a diffractometer by the addition of an area detector made the facility useful for routine data collection.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1986515

C5-116 JOURNAL DE PHYSIQUE

I1 - INSTRUMENT DESIGN

The diffractometer constructed at NBS utilized a commercial Huber model

512

goniometer mounted on a stage providing o and 28 motions. A 1 m long, 2.5 cm diameter position-sensitive detector was mounted vertically on the 28 arm of the diffractometer in a shield 35x35 cm in cross-section. The counter, manufactured in 1977 by LND (Upton, NY) was filled with 'He

(6

atm) and has worked without any failures since then. The electronic circuits have been described by Alberi et al.

/3/, with the only modification being a custom-built digital divider which replaced an analog divider described in /3/. Vertical placement of the detector gave us max- imum flexibility in crystal mounting, in that it was not necessary to align crystals along the goniometer @ axis, since rotation along any crystallographic axis could be accomplished by a combination of w , y. and @ motions of the goniometer. For reasons discussed before / 4 / , we chose flat-cone geometry of data collection in order to al- low all reflections from a given level to intersect the detector in straight lines, rather than on hyQerbolae which would result from the choice of normal-beam or equi- inclination geometries. The instrument also functions as a point-counter, single- crystal diffractometer, by applying an option to keep only the counts found near the intersection of the equatorial planes of the diffractometer and of the counter.

This mode of operation was particularly useful for filling in blind regions of the reciprocal space and for finding the initial orientation matrix.

I11

- PEAK

INTEGRATION

One of the characteristics of neutron diffraction from macromolecular crystals, especially those containing a large fraction of hydrogen atoms, is poor signal-to- noise ratio /2/. Thus it is necessary to apply such integration procedures to the reflection data which would optimize this ratio, by excluding from the peak area as many points as possible which would more properly belong to the background. A method chosen by us for this purpose was a 'dynamic mask procedure', described pre- viously /5/. This method was derived from the technique first reported by Spencer and Kossiakoff

/6/

and will be briefly summarized below.

The data measured by the detector is segregated into two-dimensional boxes, with one side composed of points measured along the detector, and the other in steps of the rotation angle 9. We assume that the position of each reflection can be predicted with an accuracy sufficient to ensure that it can fit completely within a box cen- tered at its expected position. Since the noise may prevent us from directly determining the extent of the reflection, it is necessary to apply a smoothing algo- rithm in order to suppress the noise and to emphasize the actual data. This can be done since the reflection is known to be contiguous, while the noise-is random. The steps of the dynamic mask procedure are shown schematically in Figure 1.

An actual smoothing algorithm differs depending on the application and the details, such as the number of pixels contained in a reflection box and an average count per pixel. In case of two-dimensional data such as that coming from the flat-cone dif- fractometer, we averaged each point with its eight neighbors using different weights for the central point, four nearest neighbors, and four second nearest neighbors

.

The weights found quite useful in practice were 5.2, and 1 respectively. Other choices of data and weights were necessary in one- and three-dimensional cases, which will not be discussed here.

The initial estimate of the background and of its variance can be obtained from the intensities of those points in the box unlikely to contain any parts of the peak, or on the basis of a universal background curve, which will be described below. Once the data have been smoothed, a statistical filter is applied so data belonging to the background and the peak respectively, can be distinguished. After the back- ground level and its variance have been established, the background is subtracted from each point and a flag is set, depending on the number of standard deviations by which the net intensity exceeds the background. This so-called sigma aPray also provides information for a more reliable calculation of the center of gravity for

t h e r e f l e c t i o n under i n v e s t i g a t i o n , and a t e n t a t i v e mask is s u b s e q u e n t l y o b t a i n e d by g r o u p i n g t h e c o n t i g u o u s p o i n t s o f t h e s i g m a a r r a y s t a r t i n g f r o m t h e c e n t e r o f g r a v i t y and c o n t i n u i n g o u t w a r d s . An example o f s u c h a mask is shown i n F i g u r e 2.

The Dynamb Mask AlQorltnm

d m array

Apply the parabore ~ m o o t h ~ g proseaura

Precabubte an average background n l u e and IS sigma value

c o n ~ f u c t the "sigmaanay"

Cakvlate the center of grarily bated on the rlgms array

C01)111uct the mark from the ripma an*"

111 tha canter of grarly A cyclic procedure used to flag

conldbutions Imm *her retl8slhns wnhin the Mi

C~lculnte the htepratgd IntenlHy end nr standera derlsthn

or weak reflections apply a standera mask obtained from a medium

F i g u r e 1 . Logic diagram f o r t h e dynamic mask p r o c e d u r e .

F i g u r e 2. Example o f a box c o n t a i n i n g a r e f l e c t i o n , w i t h t h e c o m p u t e d mask a n d t h e r e s u l t s of t h e i n t e g r a t i o n .

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If parts of neighboring reflections are intruding into the area used for background estimation, the resulting reflection intensity will obviously be too low. This can be detected by checking the distribution of intensities corresponding to the tenta- tive background area and removing those which differ more than 30 from the mean value. If the distribution is based on statistics only, removal of both the low and high counts will not change the average background value since the distribution is assumed to be of Poisson type and thus symmetrical. This procedure can be repeated, and we find that the background value becomes stable after, at most, three cycles of background determination.

The initial value of the background can also be estimated from the universal back- ground curve. The background value for each pixel can be estimated continuously on the basis of points falling outside reflection boxes. This background is updated by adding a fraction

f

of the current count to (1-r) of the previous count for that pixel. When a reflection has been processed, a curve (or surface) can be fitted to the universal background corresponding to the area surrounding the reflection and thus decrease the number of parameters which have to be stored from tens to only a few. Subsequently, the background for each reflection can be recomputed on the basis of the parameters describing the universal background and can be either used for the initial background estimate or combined with the local background in es- timating the final background for this reflection.

This

method

of

treating the background increases the number of points used for its estimation and thus decreases the component of the variance of the integrated intensities due to background fluctuations.

The drawback of integrating reflections using peak-search techniques lies in the danger of overestimating the intensities of those which are very weak. To prevent this from happening, the masks calculated for the weakest reflections are not used, but instead the integration is accomplished using masks from neighboring medium- strength reflections.

IV - IMPLEMENTATION OF THE PROCEDURE

Different parts of the data collection and processing procedure were implemented on two separate computers. The diffractometer itself is connected to a PDP

1 1 / 4 4 ,

which controls 10 different instruments at the NBS reactor and thus cannot be used for extensive computations. The program PSD is basically the diffractometer driver only. A crystal is mounted with the crystallographic axis which will be used for rotation located within 20' from $=O. It is possible to use less well aligned crys- tals, but some areas of the reciprocal lattice may not be accessible due to mechanical limits. An orientation matrix is computed by finding a number of strong reflections and bringing them to the equatorial plane of the instrument. The matrix is refined using the procedure of Schoemaker and Bassi /7/.

A

list of reflections for the level to be measured is prepared and is sorted according to the expected value of the angle of rotation, 6. Data are collected by step scan in

$,

usually in 0.09' increments. Each frame is divided into 512 memory locations, with each pixel corresponding to 2 mm on the detector. Counting time depends on the size of the crystal and the time available for data collection, and is usually in the range of 30 to 120 seconds.

A previously measured frame of data is processed concurrently with the counting of the current frame and the data points belonging to reflections are written into the master file, while background points are used to update the universal background ar- ray, as described above. Only limited integration is carried out on completed reflections, mainly for diagnostic purposes. Usually between one and three days are needed for a complete level of data.

Peak integration is carried out off-line on a VAX 11/780 computer. Dynamic mask

procedure as currently implemented requires two passes. In the first run only

medium strength reflections are processed in order to delineate the best average

mask, which is then used in the second pass for the weak reflections. We found that

it is not necessary in practice to apply more than one such mask, so the option in

the program enabling multiple masks is usually not utilized. In the second pass we keep track of the average offsets of reflections from the predicted positions due to an inaccurate orientation matrix. The masks are applied to the weak reflections using the positions of the centers of gravity predicted from the neighboring stronger reflections. After a complete level has been processed the shifts of the centers of gravity are used to improve the orientation matrix before the next level of data will be collected /8/. Finally, absorption correction is applied to the completed data set as described by Santoro and Wlodawer /9/. This procedure was further modified to calculate the absorption curve by scaling all data in $ bins consisting of 1 0 ' ranges, rather than by performing $ scans on individual reflections. We found that an absorption curve calculated in this manner corrects for errors due to factors other than absorption (in essence, we are performing local Scaling using JI as a variable). This type of scaling improves the agreement between multiple measurements of each reflection. Finally, we apply a correction for the influence of the incident radiation with the wavelength X/2, even though this effect was shown to be quite small / 2 / .

V -

RESULTS AND DISCUSSION

The flat-cone diffractometer has been used to collect a number of data sets from a variety of protein crystals; five data sets have been completed. Statistical infor- mation is listed in Table 1 , in the order in which these data were collected.

Fraction of reflections considered to be nobservedn in each data set is a function of the quality of the crystals, efficiency of the integration algorithm and the cut- off level. This level was set at 1.50(1) for all data except deuterated RNase,

in

which case it was set at 3o(I), eliminating a large fraction of reflections. The integration algorithm was also improved after the first data set had been collected.

hence the increase in the number of statisticalky significant reflections. The values of the R factors are higher than normally achieved in X-ray data collection, but this is primarily due to much poorer signal-to-noise ratio in neutron data col- lection, as was discussed previously (Wlodawer, 1982). It should be stressed that while the R factors were calculated using structure amplitudes F, they would be quite similar if intensities were used instead, due to the interaction between the Lorent2,factors and the integrated peak intensities, which fall off rapidly.

TABLE 1

Data Collected with a Neutron Flat-cone Diffractometer

Protein Crystal # of R Fraction

volume levels

sym Rscale

of observed RNase A, deuterated 25 16 .048-,085 .060 .51 BNase A-Uridine

vanadate, deuterated 18 22 ,039-.060 .063 .73 Insulin, deuterated 3 1 3 .lo4 .057 .60 BPTI, deuterated 8 11 .048-. 126 .076 .78 BPTI, hydrogenous 5 11 .057-.I14 . l o 8 -65

Z F-<F>

Rsy,,,=

-$&- .

calculated separately for each level except for insulin, where global value was computed.

2*Z Ff-Fe

(

RscaleP-q~;;~;~-* where Ff are structure amplitudes measured in flat-cone setting and Fe were measured using equatorial geometry.

C5-120 JOURNAL

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I n t h e most e f f i c i e n t d a t a c o l l e c t i o n procedure f o r f l a t - c o n e geometry t h e c r y s t a l s a r e mounted w i t h t h e s h o r t e s t u n i t c e l l a x i s roughly p a r a l l e l t o t h e $ a x i s of t h e d i f f r a c t o m e t e r . This c r y s t a l l o g r a p h i c a x i s is used a s an a x i s of r o t a t i o n . Such p r o c e d u r e was indeed used f o r t h e n a t i v e RNase and f o r both BPTI c r y s t a l s . I n s u l i n c r y s t a l had poorly developed f a c e s and we found i t i m p o s s i b l e t o u s e t h e s h o r t e s t (34A) h e x a g o n a l a x i s of r o t a t i o n , s o t h e 49A rhombohedra1 a x i s was used i n s t e a d . The c r y s t a l of RNase/uridine vanadate complex was wedged t h e wrong way i n t h e q u a r t z t u b e and we had t o use t h e l o n g e s t u n i t c e l l a x i s f o r r o t a t i o n , t h u s d e c r e a s i n g t h e e f f i c i e n c y of d a t a c o l l e c t i o n . This was s t i l l p r e f e r a b l e t o t h e danger of d e s t r o y -

ing t h e f r a g i l e c r y s t a l i n t h e a t t e m p t of r e o r i e n t a t i o n .

During t h e development s t a g e s of t h e dynamic mask procedure we c o n d u c t e d e x t e n s i v e t e s t s of t h e r e s u l t s of peak i n t e g r a t i o n u t i l i z i n g s t a n d a r d boxes and t h e a l g o r i t h m being developed. A s expected, we found s i g n i f i c a n t improvement due t o t h e p r o p e r c h o i c e o f t h e method of peak i n t e g r a t i o n . The improvement was seen i n t h e d e c r e a s e of t h e R f a c t o r s , i n t h e improvement of t h e I / o ( I ) r a t i o s and i n t h e i n c r e a s e of t h e f r a c t i o n of o b s e r v e d r e f l e c t i o n s . The r e a s o n s can be t r a c e d t o much improved e s - t i m a t e s of t h e background l e v e l s due t o t h e u t i l i z a t i o n of v i r t u a l l y a l l p o i n t s o u t s i d e o f t h e peaks, t o t h e i n c l u s i o n of fewer p o i n t s i n t h e a r e a s a s s i g n e d t o t h e peaks, a n d t o b e t t e r m o n i t o r i n g of t h e d e p a r t u r e of t h e c e n t e r s of g r a v i t y o f r e f l e c t i o n s from t h e p r e d i c t e d p o s i t i o n s .

The e q u a t o r i a l geometry was u s e f u l i n r e c o r d i n g d a t a from t h e b l i n d r e g i o n o f t h e f l a t - c o n e s e t t i n g , t h u s y i e l d i n g more complete d a t a s e t s . We would normally c o l l e c t d a t a i n f l a t - c o n e geometry t o t h e predetermined r e s o l u t i o n and s u b s e q u e n t l y p r o c e s s t h e s e d a t a , g e n e r a t i n g a l i s t of missing r e f l e c t i o n s . This l i s t would t h e n be used t o d r i v e t h e d i f f r a c t o m e t e r o p e r a t i n g i n t h e e q u a t o r i a l geometry. I n a d d i - t i o n , s e v e r a l h u n d r e d o v e r l a p p i n g r e f l e c t i o n s would a l s o be measured f o r s c a l i n g purposes. These d a t a would be i n t e g r a t e d u s i n g a o n e - d i m e n s i o n a l m o d i f i c a t i o n o f t h e dynamic mask procedure. The agreement of t h e d a t a c o l l e c t e d i n t h e s e two modes was q u i t e r e a s o n a b l e ( s e e Table I ) , t h u s a s s u r i n g u s of t h e p r o p e r f u n c t i o n i n g o f t h e d e t e c t o r .

The d a t a c o l l e c t e d w i t h t h e NBS neutron d i f f r a c t o m e t e r were s u c c e s s f u l l y used i n t h e refinement of s e v e r a l s t r u c t u r e s . A'model of RNase based on j o i n t X-ray and n e u t r o n refinement / I 0 / was published / I 1 /. Data from t h e R N a s e / u r i d i n e v a n a d a t e complex y i e l d e d i n t e r e s t i n g i n f o r m a t i o n a b o u t t h e p r o t o n a t i o n s t a t e s of t h e a c t i v e s i t e r e s i d u e s i n t h e enzyme /12/. S t r u c t u r e of t h e c r y s t a l form I1 of BPTI was d e t e r - m i n e d o n t h e b a s i s of t h e d a t a c o l l e c t e d by u s / 1 3 / . Hydrogen e x c h a n g e was monitored i n t h e c r y s t a l of i n s u l i n /14/ and t h e d e t a i l s of t h e s o l v e n t s t r u c t u r e i n BPTI were i n v e s t i g a t e d u s i n g d i f f e r e n c e F o u r i e r maps c a l c u l a t e d by s u b t r a c t i n g hydrogenous s t r u c t u r e amplitudes from t h e i r d e u t e r a t e d c o u n t e r p a r t s (Wlodawer, 1985.

u n p u b l i s h e d ) . F i n a l l y , we a r e now i n t h e p r o c e s s of c o l l e c t i n g n e u t r o n d i f f r a c t i o n d a t a from c r y s t a l s of DNA.

The i d e n t i f i c a t i o n of c e r t a i n commercial i n s t r u m e n t s and m a t e r i a l s does not imply recommendation o r endorsement by t h e National Bureau of S t a n d a r d s , nor does i t imply t h a t t h e m a t e r i a l s o r e q u i p m e n t i d e n t i f i e d a r e n e c e s s a r i l y t h e b e s t a v a i l a b l e f o r t h e purpose.

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