what’s new in structural biology? - x-ray...
TRANSCRIPT
03/11/20101
What’s new in Structural Biology?
Vernon SmithBruker AXS
03/11/20102
What’s new in Structural Biology at Bruker?
Vernon SmithBruker AXS
03/11/20103
Welcome to the Bio Users Meeting 2010
Thank you to our session chairs Frank von Delft, Roman Fedorov and Michael Groll
Thank you to our speakers
Thank you to our organisers Martin Adam and Anja Griessmeijer
Thank you for attending:
Czech Republic, Estonia, Finland, France, Germany, India, Ireland, Israel, Italy, Kuwait, The Netherlands, Poland, Portugal, Singapore, Slovakia, Slovenia, South Africa, Spain, Sweden, United Kingdom, USA,
03/11/20104
Users Meetings; continuing …
University of Alabama, AL S Ealick, C Smith, E Westbrook Feb. 26-29, 1989 CARB, MD G Gilliland,A Howard,G Cohen,L Hannick,A Wlodawer,M Amzel Feb. 4-6, 1990 Scripps, CA I Wilson, E Stura, D McRee, M Pique May 5-7, 1991 Purdue University, IN Wladek Minor, Ivan Rayment May 3-5, 1992 GBF Braunschweig, Germany D Schomburg, H.J.Hecht, B.Hofmann Apr. 28-30, 1993 University of Pittsburgh,PA John Rose, William Furey Apr. 28-30, 1994 University of Georgia, Athens, GA B-C Wang, John Rose Apr. 17-19, 1997 Scripps, La Jolla, CA Duncan McRee, Dave Stout Feb. 21-23, 1998 University of Bergen, Norway (ESUM98) Karl Tornroos Sep.11-13, 1998 University of Toledo, OH Alan Pinkerton June 9-11, 1999 Texas A&M University, TX Joe Reibenspies Mar. 16-17, 2000 University of Wisconsin/Bruker, WI Ilia Guzei, Charles Campana May 20-22, 2001 University of Wisconsin/Bruker, WI Ilia Guzei, Charles Campana May 18-20, 2003 University of Wisconsin/Bruker, WI Ilia Guzei Sept 20-22, 2005 Bruker AXS Delft Frank van Meurs Sept 18-19, 2006 University of Wisconsin/Bruker, WI Ilia Guzei Sept 16-18, 2007 Universität Göttingen George Sheldrick Oct 10-13, 2007 Bruker AXS Delft Frank van Meurs Sept 29-1, 2008 University of Wisconsin/Bruker, WI Ilia Guzei June 1-2, 2009 Bruker AXS Karlsruhe Martin Adam Sept 20-23, 2010
03/11/20105
Bruker News
Single crystal has moved to Bruker AXS Headquarters, Karlsruhe
New Bio Application Scientist – Dr Marianna Biadene
Strengthening service & support…
03/11/20106
Bruker AXS, Karlsruhe, Germany
Business Manager Eric Hovestreydt
Product Manager, Marketing Martin Adam
Business Development Vernon Smith
(Biological Crystallography) Bernd Hinrichsen (X2S)
Martin Adam
Vernon Smith
Eric Hovestreydt
Bernd Hinrichsen
03/11/20107
Service Karl-Heinz Bast Oliver Knaus Markus Nuesse Klaus Orth Felix Schmidt Daniel Stern
Bruker AXS, Karlsruhe, Germany
Karl-Heinz Bast Oliver Knaus Markus Nüsse
Klaus Orth Felix Schmidt Daniel Stern
03/11/20108
Bruker AXS Delft, the Netherlands
R&D Jaap Toorn
Application Leo Straver
Service Cees Baas Henk Lief Dirk Parlevliet Frank Vredenbregt
Cees Baas Henk Lief
Dirk ParlevlietFrank Vredenbregt
Leo Straver
Jaap Toorn
03/11/20109
IMCB, Singapore Manchester Interdisciplinary
Biocentre, UK University of Vienna, Austria
Protein Crystallography workshops
“… the workshop was a success and appreciated by all involved.”
(Bob Robinson, IMCB, Singapore)
03/11/201010
Product News
MX OPTICS
NEW SOURCES
X8 PROSPECTOR
IS with Quazar MX optics• No external cooling• No 3-phase power• No filament changes
Apex II detector
• High spatial resolution• Highest sensitivity• Lowest noise• Fast readout
Kappa Goniometer
• Easy sample handling• Powerful data collection tool
Source Comparison: IS
03/11/201013
• Small thaumatin crystal: 105 83 50 µm3 , = 0.5°, 120 s/°, D= 120
Resolution 2.05 Å<I/> = 12.2 (2.7)Rmerge = 0.097 (0.45)
Quazar
Resolution 1.92 Å<I/> = 13.2 (2.9)Rmerge = 0.078 (0.41)
Quazar MX
Incoatec Multilayer Optics: Comparison
X8 PROSPECTORLonger Axis at High Divergence
• Wild-Type dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase in complex with TDP
• Source Aneurinibacillusthermoaerophilus
• Beam divergence 7 mrad• Space group P41212• Unit Cell: a=b=61.7, c=201.6 Å• Hazel Holden, UW-Madison
High spatial resolution of the APEX II CCD coupled with the smaller focus of the MX optic allows separation of a longer axis even with high divergence
System Comparison: X8 PROSPECTOR
• Data were collected on the same frozen lysozyme and thaumatinsamples.
• The Rotating Anode Generator data were collected at a user site; the ImS data were collected at Bruker AXS Madison applications lab
• Match the resolution and redundancy then compare the statistics
Source RAG IS
Optics Multilayer optics
Quazar
Power load 5 kW 30 W
Detector Image plate APEXII CCD
Comparison of the IS with a conventional 5.4 kW rotating anode
Comparison Results: Thaumatin
Thaumatin crystal 1 IS RAG
Degrees collected (0.5o per frame) 270 100
Collection time (hrs) 3 9
Exposure per frame (sec) 15 60
Resolution (Å) 2.04 2.04
Rsymm 0.052 (0.13) 0.060 (0.24)
I/sigma(I) 24.7 (9.3) 11.4 (7.4)
Completeness (%) 99.1 (98.3) 98.8 (99.9)
Redundancy 6.2 6.6
Thaumatin crystal 2 IS RAG
Degrees collected (0.5o per frame) 270 210
Collection time (hrs) 3 17
Exposure per frame (sec) 15 40
Resolution (Å) 1.85 1.85
Rsymm 0.077 (0.33) 0.080 (0.69)
I/sigma(I) 14.6 (4.3) 8.5 (2.3)
Completeness (%) 99.9 (99.9) 99.9 (100)
Redundancy 5.5 7.7
In each case the X8 PROSPECTOR produced better data
• Lower Rmerge
• Higher signal to noise
• Data were collected up to 5 times faster
X8 PROSPECTOR Ligand binding studies
Resolution %Complete Redundancy Mean I/I Rmerge
Inf - 2.10 99.2 (99) 7.19 (4.9) 20.89 (3.2) 0.064 (0.40)
Data collection parameters
• Detector DX 100 mm
• Exposure time 40 sec
• Rotation angle 0.3°
• Degrees collected 540°
• Total time ~21 hrs
• Synchrotron data collected at SBC 19-BM to 1.5 Å
03/11/201018
X8 PROTEUM
High brilliance MICROSTAR rotating anode
Helios MX multi-layer optics
Platinum135 CCD detector
Kappa goniometer
Low Temp
Superb performance for
• High quality data
• Protein screening
• In-house SAD phasing
03/11/201019
NEW Turbo X-Ray Source
Compact microfocus rotating anode
Directly-mounted onto goniometer-base
= extremely stable
Simple maintenance
• Pre-aligned filaments
• Anode exchange
• Ferroseal exchange
HELIOS or HELIOS MX optics
Performance comparable with MICROSTAR
03/11/201020
Source Comparison: MICROSTAR MX
Intensity similar to 2nd generation synchrotron beamlines
03/11/201021
X8 PROTEUMHigh Resolution Data Collection
a=b=68.08 Å c=102.01 Å Source Tritirachium Crystal size 0.3x0.22x0.15 mm Exp time (sec/deg) 90 Rmerge (%) 4.1(20.4) II) 24.6(6.71) Completeness(%) 99.4(98.1) Redundancy 8.0(5.23) Collection time (hrs)40
Proteinase K at 0.95 Å
Red election density is a 2FomFc map contoured at 1, yellow density is an FomFc map at 3 showing possible hydrogen atom positions
03/11/201022
X8 PROTEUMHigh Resolution Data Collection
03/11/201023
X8 PROTEUMSulfur SAD Phasing
Source Saccharomyces cerevisiae
48 kD, 428 aa, monomer in ASU
Unit cell: a=b=85.6, c=132.8 Å,
Space group: P3121
Solvent content 56%
Substructure solved at 3.5 Å
11 Cys, 8 Met found 13 sulfurs
Redundancy ~ 50 fold to 2.5 Å
Data collected to 2.2 Å
SHELXE fit 85% of the backbone in ~30 minutes
VPS15 WD Repeat Domain
Dr. Janeen Van Hooke, Sondek lab, UNC
03/11/201024
X8 PROTEUMSulfur SAD Phasing
Structural genomics target PF1117
• Source Pyrococcus furiosus
• 85 aa, 3 met• Unit Cell: a=b=47.5, c=81.9 Å• Space group P41212
Resolution %Complete Redundancy Mean I/s R(int)
Inf ‐ 4.52 99.9 103 247.34 0.02154.52 ‐ 3.51 100 114.22 228.97 0.02733.51 ‐ 3.03 100 115.81 168.76 0.03773.03 ‐ 2.74 100 83.89 81.81 0.06232.74 ‐ 2.53 100 46.24 38.88 0.10492.53 ‐ 2.36 100 25.41 19.72 0.15882.36 ‐ 2.23 100 11.62 8.7 0.23362.23 ‐ 2.10 100 6.88 5.49 0.21882.10 ‐ 2.00 100 6.43 3.34 0.3214
Inf ‐ 2.00 100 53.56 82.84 0.0275
Data reduction statistics
B.C. Wang, University of Georgia
03/11/201025
X8 PROTEUMSulfur SAD Phasing
Substructure determination: SHELXD
•Data range 40 – 3.0 Å•3 peaks above background located
• Correlation coefficient ~50• MET23, MET68, 1 unknown
Phasing and backbone trace: SHELXE
•Phases extended to 2.0 Å•64 main chain residues found (75%)•Map generated from experimental phases contoured at 1σ•Picture shows β-strand region of molecule
03/11/201026
Sulfur SAD Phasing on X8 PROTEUMHetL, Pentapepide Repeat Protein
Source Anabaena sp.
237 resides: 4 CYS, 2 MET
Unit cell: a=68.2, b=93.7, c=101.3 Å, I222
Sub structure determined at 2.7 Åusing SHELXD
Phases were extended to 2.0 Åand the main chain atoms for all 237 residues were determined using a development version of SHELXE
Main chain fit in ~20 minutes
Structure from Mike Kennedy’s lab at Miami University of Ohio
03/11/201027
Sulfur SAD PhasingHetL, Pentapepide Repeat Protein
03/11/201028
X8 PROTEUMSAD Phasing of D-Glycerate 2-Kinase
Source E. coli 42 kD protein, 408 aa
Data resolution to 1.8 Å a=225.2,b=63.4,c=62.0 =95.6o
Crystals soaked with PtCl4, produced one heavy atom site
Overall Rsym 4.3% Average redundancy 8 fold Experimental map from one data
set collected in less than 24 hrs Chain traced by Arp/Warp overnight
03/11/201029
X8 PROTEUMUnit cell with Long Axis
P6122 a=b=70.44, c=439.6
Detector distance 200 mm Exposure time 450 sec/deg Rotation angle 0.2 Degrees collected 114 Time required ~15 hrs Divergence 3 mrad
Rmerge 6.95 (25.2) I/I 11.4 (3.2) Completeness 87% (71) Redundancy 3.0 (1.5)
Greg Petsko, Brandeis University
03/11/201030
Fast Screening30 second Lysozyme Dataset to 2.0 Å
Exposure time 0.5 seconds
Rotation width 1°Data collected 60°Total time 2.5 minutes
Total measurement time 30 seconds
Resolution range (Å) 22 - 2.00
Rmerge (%) 6.34 (17.89)
I/σ(I) 15.73 (4.24)
Completeness (%) 97.6 (85.1)
Redundancy 3.68 (1.73)
03/11/201031
Metal jet microfocus source
e-beam
liquid metalanode
x-rays
X-Ray source technology
Conventional microfocus source
solid metalanode
e-beam x-rays
Metal jet microfocus source
e-beam
liquid metalanode
x-rays
99.5 % of electron power becomes heat
Solid target close to melting
few 10 kW/mm2 define technology limit
high-speed liquid-metal-jet anode
Anode is self healing
> 500 kW/mm2
Metal jet microfocus source
e-beam
liquid metalanode
x-rays
03/11/201032
Shutter
Montel Mirror
e- optic
Metal-Jet X-ray Source (MJXS)Development model
Prototype installation, R & D Lab, Karlsruhe
03/11/201033
Target: Galinstan (eutectic mix of Ga(>75%), In, Sn) Generator Max. Energy: 60kV Kα line (Ga): 9.2keV
Development modelSource spectrum
Photon Energy
Inte
nsity
[arb
.U.]
Ga Kα 9,2 keV
Ga Kβ 10,3 keV
In Kα 24 keV
Sn Kα 25.3 keV
In Kβ 27.3 keV
Sn Kβ 28.5 keV
03/11/201034
66
1178
160
600
0 100 200 300 400 500 600 700
Intensity x 109 (X-rays/sec/mm2)
Classic 5 kW RAG/multilayer optics
IuS - Quazar
IuS - Quazar MX
EXCILLUM MJXS
MICROSTAR-H
MICROSTAR-MX
>5.0 x 1011
ph/mm2/s
X-ray SourcesComparison of flux through a 0.1 mm aperture
Beam FWHM @ sample, μm
300
240
110
260
160
~100
03/11/201035
Bruker AXS SourcesMICROSTAR
•Microfocus rotating anode generator
•Highest intensity RAG
•Dual-port
TURBO X-RAY SOURCE
•Microfocus rotating anode generator
•Compact, stable, ease-of-use
•Single-port
IS
•Microfocus sealed tube source
•Ultra reliable
•Flux greater than 5 kW RAG
METAL-JET X-RAY SOURCE
•10x brighter than current home sources
•Stability
•Low-maintenance
03/11/201036
www.bruker-axs.com