data for structural and crystallization communications - example

Example: sample preparation

At deposition, data related to preparation of sample will be accepted by RCSB in free-text form. This subsection of the data can be transmitted to the journal as part of the mmCIF file for the other sections of the data (as _pdbx_entity_prod_protocol.protocol_type and _pdbx_entity_prod_protocol.protocol) and will be included in the manuscript as is, apart from some changes in type and format. The first item can have the following values: selection, PCR, cloning, expression, growth, purification, and other. The second item will accept text input describing the preparation process identified by the first. At the journal, the contents will be evaluated in terms of the presence of data required for publication and, to help author/depositors in meeting those data requirements at time of deposition, lists of topics requiring explicit data are provided. These lists are outlines of the important experimental steps that need to be described. The text that presents these data should necessarily be brief and may be in a style of the author's choosing, but the content should be sufficient to meet the classical requirement for publication, i.e. that it permit a reader unambiguously to replicate the experiments with sufficient fidelity to be successful in achieving the published result.

In constructing the free-text description of a sample preparation from a naturally occurring source, the depositor should include specifics on the following, all of which fall under the protocol_type purification:

(1) Extraction, including as needed mechanical steps, use of solvents, delipidation and other treatments.
(2) Fractionation, including details of centrifugation, differential precipitation and resolution, chromatographic steps (volumes, concentrations, buffers, flow rates, elution protocols, temperatures), pooling of fractions and resultant volumes, estimates of yield.
(3) Other steps as needed, including, proteolytic treatments and refolding steps.
(4) Purification, including dialysis and transfer to storage buffer, composition of storage buffer, final concentration steps, protein characterization, estimate of purity, yield and method of estimation, storage temperature.

For protein production from modern artificial sources, the extraction segment should be replaced by the following, which fall into protocol_types PCR and cloning, expression and growth, and purification:

(1) Preparation of vector, including restriction enzymes digestions as needed; explicit description of the region of protein expressed with numbering referenced to a specific entry in a publicly available sequence database (e.g. NCBI RefSeq, GenBank, Swiss-Prot); PCR steps; cloning steps including methods, conditions, and details of ligation or recombination; method of transformation; modifications of vector; purification of cloned product; marker for transformed cells.
(2) Expression, including promoter; tagging procedures and sequences; volume and contents of media; incubation; details of induction, transformation or transfection; harvesting and storage of culture.
(3) Lysis, including method, composition and volume of buffer, time, temperature, and other details as necessary.

To these three segments for artificial sources should be added (as segments (4), (5) and (6) the final three segments (2), (3) and (4) listed above for natural products, with special attention being paid to specifying explicitly the retention or removal of any tags or remnants thereof and any refolding treatments that are used. If ligand must be introduced at any step during the preparation, this process should be explicitly described.

Based on Tanaka, S., Moriizumi, Y., Kimura, M. & Kakuta, Y. (2005). Overproduction, purification and preliminary X-ray diffraction analysis of a sulfotransferase from Mycobacterium tuberculosis H37Rv. Acta Cryst. F61, 33-35.

Sulfotransferase STF1 from the Mycobacterium tuberculosis H37Rv genome was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour diffusion method. The crystals diffract to 1.5Å resolution using synchrotron radiation at SPring-8. The crystals are monoclinic and belong to space group P2₁, with unit-cell parameters a = 40.86, b = 95.76, c = 48.04Å, β = 106.43°. The calculated Matthews coefficient is approximately 2.1ų Da^-1 assuming the presence of one molecule of STF1 in the asymmetric unit. A substrate-binding assay using a PAP-agarose column suggests that STF1 exhibits sulfotransferase activity.

loop_
    _pdbx_entity_prod_protocol.protocol_type
    _pdbx_entity_prod_protocol.protocol
         selection        .
         PCR
; PCR primers were designed based on the DNA sequence of the STF1 gene
  (Rv3529c) annotated from the M. tuberculosis H37Rv genome
  database. The primer sequences were 5'-primer,
  5'-ggatccATGACTCGGCGTCCCGATCGGAAA-3' (<i>Bam</i>HI site in lower case), and
  3'-primer, 5'-gaattcTCACAGCCCGGCGAACCGCTCTTT-3' (<i>Eco</i>RI site in
  lower case). The target gene was amplified by PCR using the
  following profile for 30 cycles after a preheat at 369 K: 1 min at
  369K, 30 s at 328K, 30 s at 345K (Ex Taq DNA polymerase, Takara).
;
         cloning
; The PCR product (approximately 1100 bp) was cloned into pGEM-T Easy
  (Promega) and subcloned into pGEX4T3 (Amersham Biosciences) with
  <i>Bam</i>HI and <i>Eco</i>RI sites to create a GST-STF1 fusion protein. The
  nucleotide sequence of the STF1 gene was confirmed using a CEQ
  2000KL (Beckman). The resulting expression plasmid was transformed
  into <i>E. coli</i> BL21 (DE3) Codon Plus RIL cells.
;
         expression
; The transformant was cultured in 2 x YT medium containing 100 \mg ml^-1^
  ampicillin and grown at 310 K while shaking at 200 rev min^-1^ without
  pre-culturing.
;
         growth
; After 5 h (A~600~ approx. 0.5), the culture was cooled to 293 K, kept
  for 2 h with shaking and then supplemented with 0.1 mM
  isopropyl-\b-D-thiogalactopyranoside (IPTG) for induction. After 15 h
  fermentation, the cells were harvested by centrifugation.
;
         purification
; All purifications were performed at 283 K. Harvested cells were
  suspended in a sonication buffer consisting of 50 mM Tris-HCl pH
  8.0 and 150 mM NaCl and disrupted by sonication. The lysate was
  placed in an ultracentrifuge (35,000 rev min^-1^) for 30 min at
  277 K. The harvested supernatant was gently stirred on ice with 1 ml
  glutathione Sepharose 4B affinity resin (Amersham Biosciences)
  equilibrated with sonication buffer. The resin was washed using the
  batch method with three washes of sonication buffer, two washes with
  sonication buffer containing 0.5% Triton X-100 and a final wash with
  sonication buffer. Finally, the resin with bound fusion protein was
  suspended in 5 ml sonication buffer containing 20 units thrombin
  (Sigma) and digested for 12 h at 283K. After digestion, the
  supernatant was passed through a Benzamidine Sepharose 6B (Amersham
  Biosciences) column to remove thrombin and concentrated to 0.6 mg
  ml^-1^ by ultrafiltration (Amicon Ultra-4, 10 kDa cutoff,
  Millipore). The concentrated protein was then run through a Superose
  gel-filtration column (Amersham Biosciences) using the sonication
  buffer. The flowthrough containing the target protein was collected
  and equilibrated with water by ultrafiltration. The purity of the
  prepared sample was judged using 10% SDS-PAGE stained with Coomassie
  Brilliant Blue.
;
         other
; The N-terminal sequence of the purified protein was determined with
  a PSQ-1 gas-phase sequencer (Shimadzu). The molecular weight was
  determined by gel-filtration and SDS-PAGE as described with
  chymotrypsinogen A (19.9 kDa) and ovalbumin (45.8 kDa) (Amersham
  Biosciences) used as marker proteins.
;

How this example will appear in the journal

Based on Polekhina, G., Feil, S. C., Gupta, A., O'Donnell, P., Stapleton, D. & Parker, M. W. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis. Acta Cryst. F61, 39-42.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metformin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

In the example below, the AMPK and GBD molecular species are labelled respectively as 1 and 2 in the ENTITY category that appears elsewhere in the file and is referenced by the data item _pdbx_entity_prod_protocol.entity_id.

loop_
    _pdbx_entity_prod_protocol.entry_id
    _pdbx_entity_prod_protocol.entity_id
    _pdbx_entity_prod_protocol.protocol_type
    _pdbx_entity_prod_protocol.protocol
      .    1    selection        .
      .    1    PCR              .
      .    1    cloning
;  The cloning, expression and purification of the rat AMPK\b1 subunit
   GBD (68-163) has been described previously (Polekhina <i>et al.</i>,
   2003). Briefly, GBD was cloned into pProEX HT (Invitrogen).
;
      .    1    expression
; Expressed as a His-tag fusion protein in BL21 cells.
;
      .    1    growth
; Protein expression was induced with 1 mM IPTG for 3 h at 310 K when
  the cell density reached an OD~600~ of 0.6.
;
      .    1    purification
; GBD was purified on an Ni-agarose column and precipitated with 60%
  (NH)~2~SO~4~ for 30 min at 277 K. The protein pellet was resuspended
  in 50 mM Tris-HCl pH 8.5 and desalted by gel filtration. The His tag
  was cleaved by overnight digestion at room temperature with a
  His-tagged TEV protease (Invitrogen). The TEV protease, the cleaved
  His tag and any uncleaved material were removed by a second round of
  purification on an Ni-agarose column. GBD was further purified by
  S100 gel-filtration chromatography (Amersham Pharmacia) in 50 mM
  HEPES pH 7.0. The yield of wild-type L105M GBD was 2 mg per L of
  culture.
;
      .    1    other
; This form could not be used for structure solution. Our attempts to
  determine the structure either by molecular replacement using the GBD
  model or by isomorphous replacement were unsuccessful. We therefore
  decided to introduce selenomethionine.
;
      .    1    selection
; No natural methionine residues were present in the GBD amino-acid
  sequence. Based on the GBD model and the structural alignment of
  N-isoamylase domains, Leu105 was chosen to be replaced by methionine
  since it was part of the hydrophobic core and two out of three
  N-isoamylase domains contain methionine in an equivalent position,
  while the third has a leucine.
;
      .    2    PCR
; Site-directed mutagenesis was performed according to the
  manufacturer's instructions (Stratagene). The template used was
  pProEX HT/GBD and the oligonucleotides used were
  5'-TGGAGCAAATTGCCCATGACTAGAAGCCAAAAC-3' for the sense strand and
  5'-GTTTTGGCTTCTAGTCATGGGCAATTTGCTCCA-3' for the antisense strand.
;
      .    2    cloning
; The L105M mutation was confirmed by DNA sequencing. GBD L105M was
  transformed into Novagen 834 (DE3) cells.
;
      .    2    expression
; For expression, one colony was inoculated into 3 x 100 ml minimal
  media (Molecular Dimensions) containing 40 \mg ml^-1^ methionine
  (Sigma) and incubated overnight at 310 K with shaking.
;
      .    2    growth
; The pellet washed with water was resuspended into 3 x 1 L minimal
  media (Molecular Dimensions) containing 40 \mg ml^-1^
  selenomethionine (Sigma) and incubated at 310 K with shaking until
  an OD~600~ of 0.6 was reached, at which point protein expression was
  induced by the addition of 1 mM IPTG for an additional 4 h at 310 K.
;
      .    2    purification
; The purification of SeMet-GBD L105M followed the same protocol as
  for wild-type GBD. The yield of SeMet L105M GBD was 6.7 mg per L of
  culture.
;
      .    2    other  .

The item in gray (_pdbx_entity_prod_protocol.entry_id) is an identifier needed to preserve the mmCIF data structure, and will be auto-generated during the deposition procedure.

How this example will appear in the journal

Based on C. A. A. Gadelha, C. A. A. et al. Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds. Acta Cryst. F61, 87-89.

A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 Å resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2₁2₁2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 Å. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.

loop_
    _pdbx_entity_prod_protocol.protocol_type
    _pdbx_entity_prod_protocol.protocol
         growth
; Wild mature <i>C. maritima</i> seeds were collected in the
  Cear\'a state in northeast Brazil
;
         purification
; The seeds were ground to a fine powder in a coffee mill and the
  soluble proteins were extracted at 298 K by continuous stirring with
  0.15 M NaCl [1:10 (w/v)] for 1 h, followed by centrifugation at
  10,000 g at 277K for 20 min. The supernatant was applied onto a
  Sephadex G-50 column (10 x 50 cm) previously equilibrated with 0.15
  M NaCl containing 5 mM CaCl~2~ and MnCl~2~, as described by Cavada <i>et
  al.&ly;/i> (1996). The unbound material was eluted with 0.15 M NaCl at a
  flow rate of 45 ml h^-1^ until the absorbance at 280 nm of the
  effluent stabilized at 0.05. The retained material (a lectin, called
  ConM) was eluted with 0.1 M glycine pH 2.6 containing 0.15 M NaCl,
  dialyzed exhaustively against Milli-Q water and lyophilized.
;
         other
; The purity of all ConM preparations was monitored by SDS-PAGE (Laemmli, 1970).
;

How this example will appear in the journal