The Berkeley Center for Structural Biology
The vision of the Berkeley Center for Structural Biology (BCSB) is to provide
state-of-the-art beamlines and outstanding service for crystallographers around the world, enabling structure solution on even the most complex biological systems.
For over ten years, the BCSB has operated five protein crystallography beamlines at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. Through many improvements over the years, the Center has dramatically increased the flux, stability, and automation of the beamlines, keeping them at the cutting edge of synchrotron MX science.
Two of the BCSB beamlines now have liquid nitrogen cooled monochromators,
and one of the monochromators now includes a multilayer, giving a five times flux boost for native experiments. User-controlled variable collimators (from 100 um to 10 micron spot size) are installed on four of the five beamlines, along with advanced software capabilities such as raster and vector scanning. All the beamliens are remote-enabled, significantly reducing the cost of running at the synchrotron: users need only send their crystals in pucks, and they can control the sample loading and data collection from their home labs.
The BCSB runs wiggler beamlines 5.0.1, 5.0.2, 5.0.3, and superbend
beamlines 8.2.1, and 8.2.2.
Beamlines 8.2.1 and 8.2.2 are equipped with Rigaku ACTOR robots, and the sector 5 beamlines are equipped with ALS-style robots. An MD2 microdiffractometer is installed in 8.2.1 and 8.2.2, and an MD2 has recently been installed in 5.0.2.
The successful introduction of top-off operation (constant 500mA ring current) by the ALS has resulted in a doubling in total X-ray flux delivered to users when measured across a full shift. All BCSB beamline optics have now been optimized to deliver optimal performance under 500mA operation. 5.0.1 and 5.0.3 have also had their wavelength adjusted to 12.7keV (above the Se-K edge) enabling users to use Se-SAD phasing techniques to solve their structures.
- PRT membership is now available on beamlines 5.0.1 and 5.0.2. If you are interested in trying out these beamlines, please contact Corie Ralston for more details.
- Rapid Access Proposals for structural biology beamlines are now being accepted. RAPIDD Proposals can be submitted any time, and are separate from the General User 6-month proposal cycles.
- The next ALS Shutdown will be in May/June 2014.
Recent Scientific Highlights
Chloride transport proteins can detect tiny chemical differences between ions
The CLC family of proteins are membrane proteins that control chloride intake into cells. Chloride concentration in cells is important to maintain electrical potential, as in controlling muscle contraction, and to regulate cell volume. Several congenital diseases are associated with mutations in these proteins in humans, such as Dents disease, Bartter syndrome, and osteopetrosis. One of the unique aspects of these proteins is that they are able to transport chloride ion very selectively against other anions such as fluoride and bromide, and the basis of this ability to distinguish between the different anions has been a mystery until now. But by solving a series of CLC protein structures in different ion concentrations at beamlines 8.2.1 and 8.2.2, the Miller group was able to definitively show that fluoride binds to the exact binding site in the protein as chloride. They then further determined mechanistically why fluoride halts the transport process whereas chloride doesn't. Fluoride binds specifically to the residue 148, which is known as the gating residue in the protein because it rotates into an open or closed configuration to control the flow of ions. Fluoride locks the gate in the closed position by hydrogen bonding to the carboxyl group of Gln148. This insight was made possible by the crystal structures which gave the detailed physical picture of the ion gating mechanism.
H.-H. Lim, R.B. Stockbridge, C. Miller, "Fluoride-dependent interruption of the transport cycle of a CLC Cl-/H+ antiporter", Nature Chem. Bio., v9, 721 (2013).
Ebolavirus contains a multifunctional protein that can adopt different structures depending on the needs of the virus
Ebolavirus can cause up to 90% lethality with infection. Yet despite this extremely high effectiveness, the virus is composed of only a handful of proteins. Each member of this very small team must therefore perform many different roles in the life cycle of the virus. Determining how this was possible was largely a mystery until a recent study at beamline 5.0.2 elucidated the VP40 protein in several different structural forms, including a dimer for trafficking proteins around the host cell, and a filamentous structure for forming the viral matrix. This study opens the door for designing drugs that bind to VP40 and trap it in only one of its functional states, thereby inhibiting the virus from replication.
Z.A. Bornholdt, T. Noda, D.M. Abelson, P. Halfmann, M.R. Wood, Y. Kawaoka, E.O. Saphire, "Structural REarrangement of Ebola Virus VP40 Begets Multiple Functions in the Virus Life Cycle", Cell, 154, 763 (2013).
Design of a Monoclonal Antibody with Anti-tumor activity
Onartuzumab is an example of a monovalent antibody, in that it binds its target in such a way that it does not induce dimerization of its target. In contrast, bivalent antibodies can often lead to activation rather than inhibition. Onartuzumab was designed to be specific to a particular tyrosine kinase (MET) which has been implicated in cell proliferation and tumor formation. Tradiotional bivalent antibodies were not successful in inhibiting MET, whereas onartuzumab has demonstrated anittumor activity in preclinical analysis. This paper outlined the design of this anti-cancer drug and explained its mechanism of action through crystal structures obtained at beamline 5.0.2 and biochemical analysis. An excellent movie depicting onartuzumab in action can be found here.
M. Merchant, X. Ma, H.R. Maun, Z. Zheng, J. Peng, M. Romero, A. Huang, N.-Y. Yang, M. Nishimura, J. Greve, L. Santell, Y.-W. Zhang, Y. Su, D.W. Kaufman, K.L. Billeci, E. Mai, B. Moffat, A. Lim, E.T. Duenas, H.S. Phillips, H. Xiang, J.C. Young, G.F. Vande Woude, M.S. Dennis, D.E. Reilly, R.H. Schwall, M.A. Starovasnik, R.A. Lazarus, D.G. Yansura, "Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti0tumor activity as a therapeutic agent", PNAS, Onling July 23, v.110, E2987 (2013).
An Unusual Enzyme Mechanism for Synthesizing Phosphonates
Phosphonates are compounds containing a carbon-phosphorus bond, and many naturally-occurring phosphonates have recently been found to have great use in treating human disease. This study showed how a particular enzyme is able to rearrange carbon-phosphate bonds as part of the biosynthetic pathway for producing fosfomycin, a broad spectrum antibiotic with clinical use for treating a range of infections. The crystallographic structures obtained at beamline 8.2.2 pinpointed the unique mechanism of the enzyme, and point the way for synthesizing phosphonates.
W.-C. Chang, M. Dey, P. Liu, S.O.Mansoorabadi, S.-J. Moon, Z.K. Zhao, C.L.Drennan, H.-W. Liu, "Mechanistic studies of an Unprecedented enzyme-catalysed 1,2-phosphono-migration reaction, Nature, 496, 114 (2013).
A View Through the Sugar Coating
One way that HIV hides out from antibodies is through the use of "glycan shields": sugar molecules attached to the outside of a protein that keep antibodes from recognizing and binding to the protein surface. To make matters worse, these shields are ever-changing, with the glycans moving around between residues. However, some hosts develop broadly neutralizing antibodies, which have learned to recognize the viral proteins despite the shields. Structures solved at beamline 5.0.2 show exactly how these antibodies recognize the viral surface through the glycans. In fact, the researchers discovered that one residue in particular is a "supersite" of vulnerability of HIV-1. This information can now be used to design even better antibodies to fight HIV infection.
L. Kong, J.H. Lee, K.J. Doores, C.D. Murin, J.-P. Julien, R. McBride, Y. Liu, A. Marozsan, A. Cupo, P.-J. Klasse, S. Hoffenberg, M. Caulfield, C. R. King, Y. Hua, K. M. Le, R. Khayat, M.C. Deller, T. Clayton, H. Tien, T. Feizi, R.W. Sanders, J.C. Paulson, J.P. Moore, R.L. Stanfield, D.R. Burton, A.B. Ward, I.A. Wilson, "Supersite of Immune Vulnerability of the Glycosylated Face of HIV-1 Envelope glycoprotein gp120," Nature Struct Mol Bio, 20(7), 796 (2013).
How Bacteria Defeat Antibiotics
ArcB is a membrane protein that pumps toxins out of bacteria. Unfortunately, it also pumps antibiotics out of bacterial cells, and is one of the major players in bacterial drug resistance. In this study, the structure of ArcB complexed with the antibacterial agent linezolid, an antibiotic that is often used as a "last resort" against bacterial infections, was solved at beamlines 5.0.1 and 5.0.2. The structure revealed details on the binding of the compound to the protein, and showed that the residues involved were the same as for binding of several other antibiotics such as ampicillin.
L-W. Hung, H-B. Kim, S. Murakami, G. Gupta, C-Y. Kim, and T. Terwilliger, "Crystal Structure of ArcB Complexed with Linezolid at 3.5A Resolution," J. Struct Funct Genomics, 14(2), 71 (2013).
A Portal into the Nucleus
Based on work done at beamline 8.2.1, this paper describes for the first time how the proteins that make up the nuclear pore can rearrange to dilate or constrict the pore, selectively allowing entry or exit from the nucleus by other proteins or DNA/RNA.
S.R. Solmaz, G. Blobel, and I. Melcák, "Ring cycle for dilating and constricting the nuclear pore," PNAS 110, 5858 (2013).
Cleaning Up an Environmental Toxin
PCP (pentachlorophenol) is a mjor environmental pollutant. Used in wood preservation since the 1930's, this toxic compound has worked its way into the environment where it is now a serious threat to human health. Acute contact causes convulsions and death, whereas lower exposure leads to cancer. Since clean-up through conventional means is nearly impossible, scientists have turned to specialized bacteria, which are known to break down PCP. Using beamline 8.2.1, this study elucidated the structure of the key enzyme that bacteria employ to break down the aromatic ring structure of the toxin. The structure and mechanism of catalytic activity proved unique for this enzyme, with very few similar structures ever discovered. This in itself is an interesting discovery, but in addition, by figuring out how the enzyme performs its function, this study paves the way for effective bioremediation studies.
R.P. Hayes, A.R. Green, M.S. Nissen, K.M. Lewis, L. Xun, C. Kang, "Structural characterization of 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (PcpA) from Sphingobium chlorophenolicum, a new type of aromatic ring-cleavage enzyme", Molecular Microbiology 88(3), 523 (2013).
How to Make a Diphtheria Vaccine
Diphtheria is a respiratory disease that is now rare in the industrial world, thanks to vaccines that emerged in the early part of the twentieth century. Vaccines have been extremely effective, but how do they actually work? In this study, structures from a nontoxic form of the toxin were obtained at beamline 5.0.3, showing that a single amino acid substitution makes all the difference. Despite the fact that the overall fold of the mutant and the wild-type are essentially the same, the mutant causes a change in a flexible loop near the active site that dramatically affects accessibility of the active site.
E. Malito, B. Bursulaya, C. Chen, P.L. Surdo, M. Picchianti, E. Balducci, M. Biancucci, A. Brock, F. Berti, M.J. Bottomley, M. Nissum, P. Costantino, R. Rappuoli, and G. Spraggon,
, "Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197," PNAS 109: 14, 5229 (2012)
Towards Designing a Universal Flu Vaccine
Although the annual flu is caused by a number of genetically distinct forms of the influenza viruses, there are human monoclonal antibodies that recognize and neutralize a wide range of virus forms. In this study several structures of human antibodies to these viruses were solved in part at beamline 5.0.2, and in combination with cryo-EM studies, showed how the antibodies recognize specific conserved regions across different genetic variants of viruses. The study points the way toward developing a universal flu vaccine.
C. Dreyfus, N.S. Laursen, T. Kwaks, D. Zuijdgeest, R. Khayat,
D.C. Ekiert, J.H. Lee, Z. Metlagel, M.V. Bujny, M. Jongeneelen, R. van der Vlugt, M. Lamrani, H.J.W. M. Korse, E. Geelen, Ö. Sahin, M. Sieuwerts, J.P. J. Brakenhoff, R. Vogels, O.T.W. Li, L. L. M. Poon, M. Peiris, W. Koudstaal, A. B. Ward, I.A. Wilson, J. Goudsmit, R.H.E. Friesen
, "Highly Conserved Protective Epitopes on Influenza B Viruses," Science 337, 1343 (2012)
Distinguishing Between Different Drugs Within the Same Class
Researchers at Pfizer recently solved the structures of several drug-kinase complexes from the family of drugs that are used to combat renal-cell carcinoma. These VEGR tyrosine kinase inhibitors are clinically validated and in the same drug class, yet exhibit different potencies and selectivities. The molecular structures solved at beamline 5.0.2 pinpointed drug-kinase interactions that lead to these differences, and inform future drug discovery efforts.
M. McTigue, B.W. Murray, J.H. Chen, Y.-L. Deng, J. Solowiej, R.S. Kania, "Molecular conformations, interactions, and properties associated with drug efficiency and clinical performance among VEGFR TK inhibitors," PNAS 109:45, 18281 (2012)
| || Amyloid Oligomers and Their Role in Disease |
Diseases such as Alzheimers and Parkinsons have as their hallmark aggregations of fibrous protein in plaques. However, recent evidence suggests that the cause of the aggregation is not amyloid fibrils, but rather small amyloid oligomers. Crystal structures of several amyloid oligomers solved at beamline 8.2.1 showed a cylindrical structure, and combined with biochemical studies, reinforce the theory that these oligomers are the toxic agent in amyloid diseases.
A. Laganowsky, C. Liu, M.R. Sawaya, J.P. Whitelegge, J. Park, M. Zhao, A. Pensalfini, A.B. Soriaga, M. Landau, P.K. Teng, D. Cascio, C. Glabe, D. Eisenberg, "Atomic View of a Toxic Amyloid Small Oligomer," Science 335, 1228 (2012)
| || How Proteins Keep Time |
The structures of CLOCK:BMAL1 help explain how the mammalian circadian clock is maintained: the proteins involved are transcriptional regulators that turn on protein production during the day. The same proteins that are produced as a result of this then travel into the nucleus at night and repress their own regulation. The 2.3A structure of the heterodimer delineated specific protein interfaces that stabilize the complex and allow it to function as a regulator; mutations that disturb these interfaces affect the mammalian circadian clock.
N. Huang, Y. Cheliah, Y. Shan, C.A. Taylor, S.-H. Yoo, C. Partch, C.B. Green, H. Zhang, J.S. Takahashi, "Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex," Science 337, 189 (2012)
| || A Nucleotide-Independent Voltage Gated Ion Channel |
Working through the Collaborative Crystallography program at Berkeley Center for Structural Biology, researchers at the University of Washington recently published an impressive structural analysis of a voltage-gated ion channel, proteins that control the flow of ions across a cell membrane in response to electrical potential. The structure shows that the ligand binding pocket in the C-terminal region has a negatively charged electrostatic profile, making it an unfavorable site for binding by the negatively charged nucleotides.
T.I. Brelidze, A.E. Carlson, B. Sankaran, W.N. Zagotta, "Structure of the carboxy-terminal region of a KCNH channel," Nature 481, 530 (2012)
| || TAL Effector Nuclease and its Potential Role in Genome Engineering |
TAL proteins are used by plant pathogens to target specific DNA sites within plant genes. Because they have exceptional specificity, they are considered a current prime candidate for genome engineering. Scientists from the Fred Hutchinson Cancer Research Cancer and Iowa State University solved a key TAL protein at beamline 5.0.2, providing the groundwork for which TAL proteins can be combined with endonucleases for use in targeted gene modification to combat human diseases.
A.N.-S. Mak, P. Bradley, R.A. Cernadas, A.J. Bogdanove, and B.L. Stoddard,
"The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target," Science 335, 716 (2012)
| ||Delineating the Link Between Calsequestrin and Disease |
This crystal structure of calsequestrin was the first report of specific calcium coordination sites in the protein and provided an understanding of the mechanism by which the protein binds high levels of calcium in a unique manner.
Many of the residues involved in Ca binding are found in both front-to-front and back-to-back interfaces, and are highly conserved. Mutations or binding of other ligands can interfere with the interfaces, helping explain the pathological basis for related disorders.
E.J. Sanchez, K.M. Lewis, B.R. Danna, and C. Kang, "High-capacity Ca2+-binding of human skeletal calsequestrin", JBC 287, 11592-11601 (2012)
| || Clamp-loader Complexes and DNA Replication |
A large protein complex called a "clamp-loader" is integral to DNA replication, facilitating the attachment of polymerase and the sliding clamp to DNA strands. The structure of aclamp-loader/sliding clamp complex from Bacteriophage T4 was solved at beamlines 8.2.1 and 8.2.2, leading to a detailed understanding of how the complexes move along nucleic acid.
B.A. Kelch, D.L. Makino, M. O'Donnell, and J. Kuriyan, "How a DNA polymerase clamp loader opens a sliding clamp," Science 334, 1675 (2011)
| || A Signal Recognition Particle Complex |
In co-translational protein targeting, newly translating proteins attached to the ribosome are brought to their target areas within the cell by the signal recognition particle (SRP). The 3.9A crystal structure of a prokaryotic SRP complex was solved by scientists from U.C. Berkeley and the Swiss Federal Institute of Technology using data from beamline 8.2.2.
S.F. Ataide, N. Schmitz, K. Shen, A. Ke, S. Shan, J.A. Doudna, and N. Ban, "The Crystal Structure of the Signal Recognition Particle in Complex with Its Receptor," Science 331, 881 (2011)
| || A Sesquiterpene Synthase as a Target for Biofuels |
A protein structure solved at beamlines 5.0.3, 8.2.1 and 8.2.2 has properties of unique interest to advanced biofuel production. AgBIS was solved in apo form and with several different inhibitors, showing a potential catalytic mechanism for conversion of farnesyl diphosphate into bisabolene.
R.P. McAndrew, P.P. Peralta-Yahya, A. DeGiovanni, J.H. Pereira, M.Z. Hadi, J.D. Keasling, and P. D. Adams, "Structure of a three-domain sesquiterpene synthase: a prospective target for advanced biofuels production," Structure 19, 1876-1884 (2011)
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