European Molecular Biology Laboratory (EMBL)Hamburg Outstation

by Matthias Wilmanns, Head of Outstation

Beamlines activities:

In 1998, the EMBL Hamburg Outstation has provided beam time on its sevensynchrotron radiation (SR) beam lines, covering the fields of protein crystallography,small angle scattering and X-ray absorption spectroscopy. Because of the excellentDORIS performance the beam lines were available for more than 30 weeksthroughout the year. During this time 479 projects were submitted of which 337projects (70 %) were carried out (Table 1). These numbers reflect the role of theEMBL Hamburg Outstation as one of the leading facilities of SR beam lines instructural biology. We appreciate the role of DESY to provide SR free of charge. Wethank DESY and HASYLAB for this fruitful co-operation which is widely viewed as amodel for excellent partnership.

The EMBL Hamburg Outstation has succeeded in receiving a new contract with theTMR/LSF programme of the EU for two years. With these funds we are able tosupport the access of the European users from the EU host countries to the EMBLHamburg facilities. In 1998, individual researchers from 86 different groups inAustria, Belgium, Denmark, Spain, Finland, France, Great Britain, Greece, Italy,Netherlands, Portugal and Sweden were supported. These users carried out 178projects (54 % of all projects).

Beamline instrumentation:

In 1998, the most considerable improvement was achieved at beam line X11. Theold beam line station was entirely replaced by a revised model (Figure 1) that allowsto make full use of the increased intensity that resulted from the modified cooling ofthe monochromator from 1997. The new station maintains the principle of the oldone. In this system the platform for the collimator, rotation axis of the protein crystal,crystal cooling and imaging plate detector are rotated around a point which is in thecentre of the first collimator slit pair. This system allows decoupling of translationaland rotational movements which are required for the optimisation of the photon fluxthrough the collimator. Thus, the table optimisation, required after each injection, isconsiderably facilitated.

Unfortunately, it has not yet been possible to make use of the new horizontallyfocusing double monochromator system on the wiggler beam line. Extensive testingof this system on beam line X12 led to a number of modifications and improvements.During the last section of beam time the monochromator was built in. The tests ofthe monochromator have to be finalised at the beginning of the beam time in 1999.

A description by Roelof van Silfhout on the new water-cooled monochromator on thewiggler beam line BW7B can be found in the technical section of the HASYLABreport.

Table 1: Summary of Users, Groups and Projects 1998

Country Individualusers

UserGroups

Projectsperformed

EU member and associated states:AustriaBelgiumGermanyDenmarkSpainFinlandFranceUnited KingdomGreeceItalyNetherlandsPortugalSweden

15 31 217 13 19 5 29 113 9 25 32 3 22

2 7 63 2 5 2 9 30 2 8 6 2 7

6 16105 7 19 5 14 68 3 15 18 2 20

Total 533 145 298Other European states:

BulgariaPolandRussiaSlovakiaSwitzerland

1 1 1 1 14

2 1 1 2 7

4 1 1 4 15

Total 18 13 25Non-European states:IndiaCanadaNew ZealandUSA

1 4 1 8

1 2 1 4

1 5 1 7

Total: 14 8 14Grand Total 565 166 337

Users from EU member states and associated states are eligible for support oftravel and accommodation. Please consult the EMBL Hamburg Internet pages forfurther restrictions: (http://www.embl-hamburg.de/tmr/index.html).

Course and workshop programme:

In October 1998 an advanced training course on the expression, purification andcrystallisation of proteins was organised by Paul Tucker (chair), Ingeborg Feil,Matthias Wilmanns and Eila Cedergren-Zeppezauer (Lund). From 140 applicationsonly 16 could be accepted. This number demonstrates the strong demand in thistype of course programme. We thank all speakers, tutors and participants for theiroutstanding contributions. This course was supported by the EU TMR/LSF contract.

An exploratory workshop entitled “Signal transduction by phosphorylation in the cell:integrating structure and function”, was organised by Matthias Wilmanns and GiulioSuperti-Furga from EMBL Heidelberg. The workshop brought leading experts fromgenetics, cell biology, biochemistry and structural biology together. The workshopwas supported by ESF.

Figure 1: the new experimental station of beamline X11

(1) Mar imaging plate, (2) Video microscope, (3) collimator with integrated ionisationchambers, (4) cardanic mounting, (5) beam exit, (6) detector translation with integrated ruler(precision 1/100 mm), (7) optical table ('honeycomb'), (8) aluminium frame structure onwheels, (9) rotation axis for protein crystal with an integrated angle measuring device(precision 1/1000 deg.).

Outlook to 1999

During this year beamline X13, previously used by the muscle and lipids group, willbe converted into a protein crystallography beamline. The set-up of the beam linewill be similar to that of X11. The aim is to make this beam line available to externalacademic and industrial investors. At least 1/3 of the overall time will be available tothe general user community. Strong focus by the instrumentation group will be alsoon the upgrading of the tuneable beam line X31 and on finalising the opticalelements of BW7A.

In protein crystallography two new staff scientists, Alexander Popov and EhmkePohl, have started at the begiining of this year. Paul Tucker, group leader in proteincrystallography, will be available during most of the beam time.

The Outstation is planning the following courses or workshops in 1999:

1. Potential Applications in Structural Biology of an X-ray Free Electron Laser atDESY, April 1999, organised by Matthias Wilmanns, Victor Lamzin and Prof.Jochen Schneider from HASYLAB.

2. X-ray data collection, processing and phasing, October/November, organised by

Victor Lamzin et al. 3. EXAFS data collection and processing, June, organised by Wolfram Meyer-

Klaucke and Paula d’Angelo (Rome). 4. Shape determination and envelope functions, organised by Michel Koch and

Dimitri Svergun. New proposals:

Beam proposal forms are available through the WWW home page of the EMBLOutstation (http://www.embl-hamburg.de). Further information can be obtained byphone: +49-40-89902-0, by fax: +49-40-89902-149, or by Email: beam@embl-hamburg.de (protein crystallography), koch@embl-hamburg.de (small anglescattering of non-crystalline systems and wolfram@embl-hamburg.de (X-rayabsorption spectroscopy).

Scientific support staff:Christiane Blessing (computer), Thomas Eriksson (computer), Ingeborg Feil (wetlaboratory), Ana Gonzales (protein crystallography), Christoph Hermes(instrumentation), Michel Koch (non-crystalline systems), Victor Lamzin (proteincrystallography), Wolfram Meyer-Klaucke (X-ray absorption spectroscopy),Alexander Popov (protein crystallography), Gerd Rapp (muscle and lipids), WoytekRypniewski (protein crystallography), Dimitri Svergun (non-crystalline systems),Paul Tucker (protein crystallography), Roelof Van Silfhout (instrumentation),Matthias Wilmanns (protein crystallography).

Technical and administrative support staff:

Peter Bendall (computer), Santiago Canela (beam line assistance), Margret Fischer(administration), Graham Frost (beam line assistance), Thomas Gehrmann

(instrumentation), Sabine Gemeinhardt (wet lab), Jens Hartmann (instrumentation),Roy Klaering (instrumentation), Niek Kunst (muscle & lipids), Rika Pfaff(administration), Bernd Robrahn (Instrumentation), Viktor Renckwitz(instrumentation), Heinz-Dieter Genz (care taker).

EMBL staff

Scientifc Priorities Committee :

Martino Bolognesi (Genova, Italy; chair of the Priorities Committee), Zbigniew Dauter(Brookhaven, USA), Phil Evans (Cambridge, UK), Jean Doucet (Paris, France),Roger Goody (Dortmund, Germany), Jose Goulon (Grenoble, France), UdoHeinemann (Berlin, Germany), Stefano Mangani (Siena, Italy), Kyriakos Petratos(Heraklion, Greece), Gunter Schneider (Stockholm, Sweden), Joel Sussman(Rehovot, Israel).

Other sources for further information about the EMBL Hamburg activities :

1. Internet: htttp://www.embl-hamburg.de

2. EMBL Annual Report

3. EMBL Research Report

4. EMBL Hamburg Outstation report

Protein Crystallography on the MPG/GBF Beamline BW6

The wiggler beamline BW6 at DORIS has been fully dedicated to protein crystallography in thebeginning of 1997. The facility has been used primarily by groups from the Max-Planck Society(MPG) and the Society of Biotechnological Research (GBF). It has further been used by universitygroups from Europe and the USA, and by pharmaceutical industry. Prospective users are invited tovisit the webpage www.mpasmb-hamburg.mpg.de for additional information on the beamline andfor applying for beamtime. Rapid access is possible for urgent competitive projects. For furtherinformation on possible applications and collaborative work contact Hans D. Bartunik(bartunik@mpghdb.desy.de).

MAD phasingBW6 is optimized for rapid experimental phasing of protein structures using MAD methods. Theequipment for tuning the X-ray wavelengths, X-ray fluorescence scans, diffraction measurements(using CCD or image-plate detectors), and computing together with software installations permitsinteractive assessment of the feasibility of MAD phasing and phase evaluation. The aim to obtaininterpretable initial Fourier maps within a few days after starting data collection has been achievedin a number of applications. The possibility to solve a new structure by MAD methods using onesingle crystal only was essential in particular for the crystal structure analysis of an integralmembrane protein, ba3 cytochrome-c oxidase from T. thermophilus, at 2.0 Å resolution (Than etal.). The relatively small anomalous effects at the K-edges of Fe and Cu were sufficient to phasethis structure of 80 kD. Two different anomalous scatterers, Pt and Zn, were used also in the MADphasing of pitrilysin (Maskos et al.); this structure contains one monomer of 110 kD perasymmetric unit. Further applications are described in the user reports.

High-MW structuresThe work by Ada Yonath and her group progressed substantially towards a crystal structuresolution of ribosomes (Bartels et al.). In another top priority project on the beamline, the structure-function relationships of the 20S proteasome from yeast were investigated on the basis of high-resolution crystal structures of mutants and complexes with inhibitors (M.Groll et al.). Thisheterogeneous multi-protein system has a total of 700 kD per asymmetric unit.

Ultra-high resolution studiesCrystal structure analyses of heme proteins at atomic resolution (Popov et al.) showed that changesin ligandation require concerted motions of the heme and the protein matrix involving long-rangecorrelations. These motions were not visible in previous studies at lower resolution.

CCD detectorThe use of a MAR CCD detector system substantially reduced the total time required perexperiment, due to short read-out times. The overall signal-to-noise and the quality of thediffraction data compare favourably with a MAR345 image-plate scanner which is also availableon BW6; the quantum detection efficiencies are similar. The short "dead time" with the CCDsystem made it feasible in a number of applications to collect data to high resolution despite largecrystal mosaicity limiting the maximum rotation range per exposure to, e.g., 0.1 . Rapid changefrom one detector system to the other is achieved under computer control. Complete diffractiondata sets may be collected in an unattended automatic mode including continuous realignment ofthe beamline.

Beamline characteristicsBW6 is a wiggler beamline with double-focussing X-ray optics providing a relatively small beamcross-section at the focus, due to (3.3:1) demagnifying imaging. At the same time, a relatively longdistance (10 m) of the focusing toroidal mirror to the focal plane results in fine collimation. As aconsequence, structures with cell dimensions > 2000 Å may be investigated. The beamline can beoperated under monochromatic conditions (using a Si(111) double-crystal monochromator) withrapid tunability over the wavelength range 0.6 < λ < 2.0 Å, or under white-beam conditions(> 0.55 Å). Laue diffraction is feasible on time scales ranging from nanoseconds to seconds;facilities for optical excitation and spectroscopy can be provided. The beamline is equipped with aCRYOSTREAM (Oxford Cryosystems) for crystal cooling to 90 K. Biochemical lab facilitiesmay be used for sample preparation or crystallization. Several UNIX workstations are available forimmediate data processing, inspection of electron density maps, and complete crystal structureanalysis.