high-speed macromolecular structure determination on a superbend beamline 8.3.1 j.m. holton 1, c....
TRANSCRIPT
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High-speed macromolecular structure determination on a SuperbendBeamline 8.3.1
J.M. Holton1, C. Chu2, K. Corbett2, J. Erzberger2, R. Fennel-Fezzie2, J. Turner3 , D. Minor3 , R.J. Fletterick3 , J.M. Berger2, T.C. Alber2
1Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, Berkeley, CA, 3University of California, San Francisco, CA
The work was performed at the Advanced Light Source of Lawrence Berkeley National Laboratory, which is operated by
Departments of Energy’s Office of Basic Energy Science with Contract No. DE-AC03-76SF00098.
ElvesMOSFLM SCALA TRUNCATE SCALEIT SHELX SOLVE MLPHARE DM ARP_WARP REFMAC
Drug Discovery
UnderstandingDisease
New insights
Primase (DnaG) proteins initiate the DNA replication process in all forms of life. This S. aureus primase was solved to 1.8Å resolution at 8.3.1 and illustrates the high degree of conservation in the structure of this molecule in every living thing.
DNA replication initiation
Superbend
Parabolic mirror
Torroid mirror
Si(111) monochromator
Protein Crystal(preserved at 90K
in nylon loop)
Diffraction Images(~1000)
Atomic Model(1000-1,000,000 atoms)
Electron density at 3.5Å from a bacterial chromosome condensation and segregation protein. Two -helices are apparent and two selenium atom positions are shown in green. This initial map was obtained less than one hour after the data collection began.
Chromasome condensation
The structure of this bacterial DNA Replication initiation protein (DnaA) suggests a common structural theme in replication initiation across all kingdoms of life. This structure was solved to 2.7Å resolution at ALS Beamline 8.3.1 in less than one hour.
Electron density at 2.5Å from a DNA topoisomerase subunit. This enzyme untangles DNA molecules during replication. This section of density highlights an isolated -helix.
DNA topology
Electron density at 1.5Å from a designed protein. This new protein was conceived using structural information from dozens of natural proteins. The high resolution structure validates our understanding of how natural proteins specify their structures. This map was obtained five hours after the data collection began.
Protein design
MCAK protein strongly resembles motor proteins that crawl along microtubules (kinesins). However, MCAK actively depolymerizes microtubules in the kinetochore. The structure of MCAK helps us understand how similar structures can have radically different functions.
Protein motors
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What is Protein?
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What is Protein?
• 50% (dry weight) of cells
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What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans
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What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans
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What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans• Large molecules (1000-1000000 atoms)
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What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans• Large molecules (1000-1000000 atoms)
• Incredibly well-organized
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What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans• Large molecules (1000-1000000 atoms)
• Incredibly well-organized
• All 30,000 necessary for life
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What do Proteins do?
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What do Proteins do?
• Break down food
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What do Proteins do?
• Break down food
• Build new molecules
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What do Proteins do?
• Break down food
• Build new molecules
• Hold cells together
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What do Proteins do?
• Break down food
• Build new molecules
• Hold cells together
• Move objects
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Aspartate Transcarbamoylase
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Proteins Move
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How do you get the structure?
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How do you get the structure?
• Purify the protein
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How do you get the structure?
• Purify the protein
• Crystallize it
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
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Protein Expression
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Protein Expression
genePCR
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Protein Expression
genePCR
E. coli
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Protein Expression
genePCR
plasmid E. coliDNA
extract
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Protein Expression
genePCR
plasmid
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Protein Expression
genePCR
plasmidcut
plasmid
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Protein Expression
genePCR
plasmidrecombinant
plasmid
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Protein Expression
genePCR
plasmidrecombinant
plasmidE. coli
transform
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Protein Expression
genePCR
plasmidrecombinant
plasmidE. coli
E. coliE. coli
growth
transform
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Protein Expression
E. coliE. coli
lysis
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Protein Purification
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Protein Purification
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How much do proteins cost?
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How much do proteins cost?
• Gold: $450/ounce
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How much do proteins cost?
• Gold: $450/ounce
• Lysozyme: $18,000/ounce
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How much do proteins cost?
• Gold: $450/ounce
• Lysozyme: $18,000/ounce
• HIV protease: ~$109/ounce
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How much do proteins cost?
• Gold: $450/ounce
• Lysozyme: $18,000/ounce
• HIV protease: ~$109/ounce
• Antimatter: ~$1015/ounce
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Protein Purification
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
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Protein Purification
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Crystallize it
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Crystallize it
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Crystallize it
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Crystallize it
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Crystallize it
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
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Mount The Crystal
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Mount The Crystal
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Mount The Crystal
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Zero-parallax optics
pinhole
prism
microscope
backstop
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Zero-parallax optics
pinhole
prism
microscope
backstop
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Zero-parallax optics
pinhole
prism
microscope
Styrofoam™ backlight
backstop
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Zero-parallax optics
pinhole
prism
microscope
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
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Electron-density map
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How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
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Build an atomic model
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Build an atomic model
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Build an atomic model
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Meaning of “resolution”
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Meaning of “completeness”
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Meaning of “phase”
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High-speed macromolecular structure determination on a SuperbendBeamline 8.3.1
J.M. Holton1, C. Chu2, K. Corbett2, J. Erzberger2, R. Fennel-Fezzie2, J. Turner3 , D. Minor3 , R.J. Fletterick3 , J.M. Berger2, T.C. Alber2
1Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, Berkeley, CA, 3University of California, San Francisco, CA
The work was performed at the Advanced Light Source of Lawrence Berkeley National Laboratory, which is operated by
Departments of Energy’s Office of Basic Energy Science with Contract No. DE-AC03-76SF00098.
ElvesMOSFLM SCALA TRUNCATE SCALEIT SHELX SOLVE MLPHARE DM ARP_WARP REFMAC
Drug Discovery
UnderstandingDisease
New insights
Primase (DnaG) proteins initiate the DNA replication process in all forms of life. This S. aureus primase was solved to 1.8Å resolution at 8.3.1 and illustrates the high degree of conservation in the structure of this molecule in every living thing.
DNA replication initiation
Superbend
Parabolic mirror
Torroid mirror
Si(111) monochromator
Protein Crystal(preserved at 90K
in nylon loop)
Diffraction Images(~1000)
Atomic Model(1000-1,000,000 atoms)
Electron density at 3.5Å from a bacterial chromosome condensation and segregation protein. Two -helices are apparent and two selenium atom positions are shown in green. This initial map was obtained less than one hour after the data collection began.
Chromasome condensation
The structure of this bacterial DNA Replication initiation protein (DnaA) suggests a common structural theme in replication initiation across all kingdoms of life. This structure was solved to 2.7Å resolution at ALS Beamline 8.3.1 in less than one hour.
Electron density at 2.5Å from a DNA topoisomerase subunit. This enzyme untangles DNA molecules during replication. This section of density highlights an isolated -helix.
DNA topology
Electron density at 1.5Å from a designed protein. This new protein was conceived using structural information from dozens of natural proteins. The high resolution structure validates our understanding of how natural proteins specify their structures. This map was obtained five hours after the data collection began.
Protein design
MCAK protein strongly resembles motor proteins that crawl along microtubules (kinesins). However, MCAK actively depolymerizes microtubules in the kinetochore. The structure of MCAK helps us understand how similar structures can have radically different functions.
Protein motors